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Sample records for allele-specific loop-mediated amplification

  1. Detection of mutation by allele-specific loop-mediated isothermal amplification (AS-LAMP).

    PubMed

    Aonuma, Hiroka; Badolo, Athanase; Okado, Kiyoshi; Kanuka, Hirotaka

    2013-01-01

    For effective control of pathogen-transmitting mosquitoes, precise surveillance data of mosquito distribution are essential. Recently, an increase of insecticide resistance due to the kdr mutation in Anopheles gambiae, a mosquito that transmits the malaria parasite, has been reported. With the aim of developing a simple and effective method for surveying resistant mosquitoes, LAMP was applied to the allele-specific detection of the kdr gene in An. gambiae. Allele-specific LAMP (AS-LAMP) method successfully distinguished the kdr homozygote from the heterozygote and the wild type. The robustness of AS-LAMP suggests its usefulness for routine identification of insects, not only mosquitoes but also other vectors and agricultural pests. Here we describe the method of AS-LAMP to detect mutation in Anopheles mosquitoes. PMID:24026691

  2. Loop-Mediated Isothermal Amplification (LAMP) Signature Identification Software

    2009-03-17

    This is an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs.

  3. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    PubMed

    Poole, Catherine B; Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C; Carlow, Clotilde K S

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis. PMID:23272258

  4. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    PubMed

    Poole, Catherine B; Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C; Carlow, Clotilde K S

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  5. Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    PubMed Central

    Poole, Catherine B.; Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C.; Carlow, Clotilde K. S.

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis. PMID:23272258

  6. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    PubMed

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical.

  7. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    PubMed

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical. PMID:22327143

  8. Determination of DQB1 alleles using PCR amplification and allele-specific primers.

    PubMed

    Lepage, V; Ivanova, R; Loste, M N; Mallet, C; Douay, C; Naoumova, E; Charron, D

    1995-10-01

    Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

  9. Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an in...

  10. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

    PubMed Central

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-01-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease. PMID:27284221

  11. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

    PubMed

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease. PMID:27284221

  12. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    SciTech Connect

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. )

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  13. Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP).

    PubMed

    Zhang, Xuzhi; Lowe, Stuart B; Gooding, John Justin

    2014-11-15

    The loop-mediated isothermal amplification (LAMP) technique has the potential to revolutionize molecular biology because it allows DNA amplification under isothermal conditions and is highly compatible with point-of-care analysis. To achieve efficient genetic analysis of samples, the method of real-time or endpoint determination selected to monitor the biochemical reaction is of great importance. In this paper we briefly review progress in the development of monitoring methods for LAMP.

  14. A Novel Real-Time DNA Detection System for Loop-Mediated Isothermal Amplification Method

    NASA Astrophysics Data System (ADS)

    Kakugawa, Koji; Yamada, Kenji; Maeda, Hiroshi; Takashiba, Shougo

    We developed a novel real-time DNA detection system for loop-mediated isothermal amplification (LAMP) method. Our prototype was composed of a thermostatic chamber, a hole slide glass, LED and a web camera. The reaction mixture was injected into the slide glass hole and the LAMP reaction was carried out at 63°C for 2 hours. To observe the DNA amplification, we monitored the fluorescence intensity of SYBR Green I that was excited by the blue LED. The captured BMP images were analyzed by NIH Image J software. The DNA amplification and amplification monitoring experiment was successful. Furthermore, quantitative accuracy was evaluated based on real-time PCR. The reaction time correlates well with the DNA concentration. These results indicate the successful development of a novel real-time DNA detection system for LAMP method.

  15. Loop-mediated isothermal amplification (LAMP) for the rapid detection of Mycoplasma genitalium.

    PubMed

    Edwards, Thomas; Burke, Patricia; Smalley, Helen B; Gillies, Liz; Longhurst, Denise; Vipond, Barry; Hobbs, Glyn

    2015-09-01

    Mycoplasma genitalium is a sexually transmissible, pathogenic bacterium and a significant cause of nongonococcal urethritis in both men and women. Due to the difficulty of the culture of M. genitalium from clinical samples, the laboratory diagnosis of M. genitalium infection is almost exclusively carried out using nucleic acid amplification tests. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, utilising a set of 4 primers specific to 6 distinct regions of the target DNA sequence, in order to amplify target DNA in a highly specific and rapid manner. A LAMP assay was designed to the pdhD gene of M. genitalium, and the limit of detection of the assay was determined as 10 fg of M. genitalium genomic DNA, equating to ~16 copies of the M. genitalium genome, which was equally sensitive as a gold standard 16S rRNA polymerase chain reaction assay. PMID:26072150

  16. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants

    PubMed Central

    Lee, Siwon; Kim, Jin-Ho; Choi, Ji-Young; Jang, Won-Cheoul

    2015-01-01

    We developed a loop-mediated isothermal amplification (LAMP) method to rapidly diagnose Wheat streak mosaic virus (WSMV) during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections. PMID:26674930

  17. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants.

    PubMed

    Lee, Siwon; Kim, Jin-Ho; Choi, Ji-Young; Jang, Won-Cheoul

    2015-12-01

    We developed a loop-mediated isothermal amplification (LAMP) method to rapidly diagnose Wheat streak mosaic virus (WSMV) during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections. PMID:26674930

  18. Monitoring the progression of loop-mediated isothermal amplification using conductivity.

    PubMed

    Zhang, Xuzhi; Liu, Wenwen; Lu, Xun; Justin Gooding, J; Li, Qiufen; Qu, Keming

    2014-12-01

    Loop-mediated isothermal amplification (LAMP) yields a large amount of DNA, as well as magnesium pyrophosphate precipitate, causing a decrease in ionic strength that can be measured with a conductivity meter. There is a clear relationship between the conductivity of the LAMP mixture solution and the duration of biochemical reaction. Moreover, there is also a clear relationship between the change in conductivity and the amount of initial template DNA over the range of 0.08 to 3.2 ng. These results demonstrate the feasibility not only for detecting the LAMP product qualitatively but also for real-time monitoring the biochemical reaction progression quantitatively using conductivity measurements.

  19. Visual detection of murray valley encephalitis virus by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Gong, Rui; Wang, Han Hua; Qin, Hong; Guo, Xiao Ping; Ma, Xue Jun

    2015-03-01

    A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 °C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV. PMID:25800449

  20. Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification.

    PubMed

    Ren, Chun-Hua; Hu, Chao-Qun; Luo, Peng; Wang, Qing-Bai

    2009-01-01

    Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 degrees C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.

  1. Loop-mediated isothermal amplification method for a differential identification of human Taenia tapeworms.

    PubMed

    Sako, Yasuhito; Nkouawa, Agathe; Yanagida, Tetsuya; Ito, Akira

    2013-01-01

    Loop-mediated isothermal amplification (LAMP), which employs a Bst DNA polymerase with strand-displacement activity and four primers (two inner primers and two outer primers) recognizing six distinct regions on the target DNA, is a highly sensitive, specific, simple, and rapid nucleotide amplification method. Moreover, because the Bst DNA polymerase resists much DNA polymerase inhibitors present in biological specimens, the LAMP method is suitable for the detection of infectious agents from clinical material such as fecal samples. Here, we describe the LAMP method which can differentially detect and identify human Taenia tapeworms, Taenia solium, T. saginata, and T. asiatica, using DNA specimens prepared from parasite tissue and human fecal sample. PMID:24026690

  2. Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii

    PubMed Central

    Wang, Qinqin; Zhou, Yanbin; Li, Shaoli; Zhuo, Chao; Xu, Siqi; Huang, Lixia; Yang, Ling; Liao, Kang

    2013-01-01

    Background Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii. Methodology and Significant Findings Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively. Conclusion The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has

  3. Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

    NASA Astrophysics Data System (ADS)

    Meng, Qinglei; Wang, Shi; Zhang, Lingling; Huang, Xiaoting; Bao, Zhenmin

    2013-01-01

    In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop ( Chlamys farreri).

  4. [Visual Detection of Human Coronavirus NL63 by Reverse Transcription Loop-Mediated Isothermal Amplification].

    PubMed

    Geng, Heyuan; Wang, Shengqiang; Xie, Xiaoqian; Xiao, Yu; Zhang, Ting; Tan, Wenjie; Su, Chuan

    2016-01-01

    A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63. PMID:27295884

  5. Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification.

    PubMed

    Ahmed, Minhaz Uddin; Saito, Masato; Hossain, M Mosharraf; Rao, S Ramachandara; Furui, Satoshi; Hino, Akihiro; Takamura, Yuzuru; Takagi, Masahiro; Tamiya, Eiichi

    2009-05-01

    In this study, we are reporting for the first time an efficient, accurate and inexpensive rapid detection system which employs the integration of isothermal amplification and subsequent analysis of unpurified amplicons by an electrochemical system. In our experiments, loop-mediated isothermal amplification (LAMP) with its higher efficiency than PCR was performed at a constant temperature (65 degrees C). Amplification products were combined with a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)] and analyzed by a DNA stick (DS) which is integrated with a disposable electrochemical printed (DEP) chip using linear sweep voltammetry (LSV). The DNA minor groove binding of the H33258 molecule causes a significant drop in the peak current intensity of the H33258 oxidation. The phenomenon of DNA binding induced by H33258, in addition to changes in the anodic current peak, was used to detect maize CBH 351 variety (StarLink). Since laborious probe immobilization was not required, and amplification and detection were performed on a single device, our biosensor eliminates potential cross-contamination. We believe that this type of sensor will have an unprecedented impact for environmental protection.

  6. Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification.

    PubMed

    Ahmed, Minhaz Uddin; Saito, Masato; Hossain, M Mosharraf; Rao, S Ramachandara; Furui, Satoshi; Hino, Akihiro; Takamura, Yuzuru; Takagi, Masahiro; Tamiya, Eiichi

    2009-05-01

    In this study, we are reporting for the first time an efficient, accurate and inexpensive rapid detection system which employs the integration of isothermal amplification and subsequent analysis of unpurified amplicons by an electrochemical system. In our experiments, loop-mediated isothermal amplification (LAMP) with its higher efficiency than PCR was performed at a constant temperature (65 degrees C). Amplification products were combined with a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)] and analyzed by a DNA stick (DS) which is integrated with a disposable electrochemical printed (DEP) chip using linear sweep voltammetry (LSV). The DNA minor groove binding of the H33258 molecule causes a significant drop in the peak current intensity of the H33258 oxidation. The phenomenon of DNA binding induced by H33258, in addition to changes in the anodic current peak, was used to detect maize CBH 351 variety (StarLink). Since laborious probe immobilization was not required, and amplification and detection were performed on a single device, our biosensor eliminates potential cross-contamination. We believe that this type of sensor will have an unprecedented impact for environmental protection. PMID:19381392

  7. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis.

    PubMed

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis. PMID:24792836

  8. Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

    PubMed

    Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

    2013-11-01

    The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

  9. Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.

    PubMed

    Mirzai, S; Safi, S; Mossavari, N; Afshar, D; Bolourchian, M

    2016-01-01

    The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders. PMID:27609471

  10. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis.

    PubMed

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis.

  11. Loop-mediated isothermal amplification (LAMP)-based method for rapid mushroom species identification.

    PubMed

    Vaagt, Franziska; Haase, Ilka; Fischer, Markus

    2013-02-27

    Toxic mushroom species, such as the death cap ( Amanita phalloides ), are responsible for most mushroom poisonings. In the present work, novel loop-mediated isothermal amplification (LAMP) assays were used for the differentiation of even closely related edible and toxic mushroom species. The applicability of these methods was tested by cross-reaction studies and analysis of spiked mushroom samples (raw and fried material). Contaminations at the level of 2% (w/w) could be detected in different mushroom blends. Three detection methods were used: agarose gel analysis, fluorimetric real-time detection, and visual detection by lateral flow dipsticks (LFD). The LAMP assay combined with LFD detection allows the identification of A. phalloides in about 2 h (including DNA extraction) at a very low level of technical equipment (micropestle, water bath, and mobile centrifuge), which makes this technique perfectly suited for on-site applications.

  12. Detection of acute toxoplasmosis in pigs using loop-mediated isothermal amplification and quantitative PCR.

    PubMed

    Wang, Yanhua; Wang, Guangxiang; Zhang, Delin; Yin, Hong; Wang, Meng

    2013-10-01

    A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

  13. A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification.

    PubMed

    Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takada, Aya; Shojo, Hideki; Adachi, Noboru; Saito, Kazuyuki

    2011-01-01

    We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice. PMID:21198609

  14. Development of reverse transcription loop mediated isothermal amplification assay for rapid detection of bluetongue viruses.

    PubMed

    Mohandas, Sreekala S; Muthuchelvan, Dhanavelu; Pandey, Awadh Bihari; Biswas, Sanchay Kumar; Chand, Karam; Venkatesan, Gnanavel; Choudhary, Dheeraj; Ramakrishnan, Muthannan Andavar; Mondal, Bimalendu

    2015-09-15

    A single-step reverse transcription loop mediated isothermal amplification (RT-LAMP) assay targeting NS1 - a highly conserved gene among BTV serotypes was optimized and validated with seven serotypes: BTV-1, BTV-2, BTV-9, BTV-10, BTV-16, BTV-21 and BTV-23. The relative sensitivity of the assay was 0.3 TCID50 and no cross reactivity could be observed with foot and mouth disease, peste-des-petits-ruminants, goatpox, sheeppox and orf viruses. The established assay was also assessed by screening of clinical samples and the result is comparable with conventional RT-PCR. The RT-LAMP assay described here could be an additional tool to the existing assays for diagnosis/surveillance of BTV. PMID:26073661

  15. Real-time fluorescence loop-mediated isothermal amplification for the diagnosis of hemorrhagic enteritis virus.

    PubMed

    Liu, Xuemei; Li, Yuhao; Xu, Chenggang; Qin, Jianru; Hao, Jianyong; Feng, Min; Tan, Liqiang; Jia, Weixin; Liao, Ming; Cao, Weisheng

    2014-04-01

    Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/μl, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research.

  16. Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia.

    PubMed

    Lau, Yee-Ling; Lai, Meng-Yee; Fong, Mun-Yik; Jelip, Jenarun; Mahmud, Rohela

    2016-02-01

    The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent.

  17. An improved loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis

    PubMed Central

    HIGA, Yumiko; UEMURA, Ryoko; YAMAZAKI, Wataru; GOTO, Shinya; GOTO, Yoshitaka; SUEYOSHI, Masuo

    2016-01-01

    We improved a loop-mediated isothermal amplification (LAMP) assay permitting sensitive and rapid Mycoplasma bovis detection. A total of 55 bacterial strains were examined in this study, including 33 M. bovis strains, 14 non-M. bovis mycoplasmas and eight non-mycoplasma bacterial strains. M. bovis was successfully detected by the LAMP assay within 60 min without cross-reaction to any other bacteria. Furthermore, a total of 135 nasal swab samples were tested directly using our LAMP assays, the previously reported LAMP assay, conventional PCR assay without pre-culture and comparing standard culture methods. The improved LAMP assay showed sensitivity and specificity of 97.2% and 90.9%, respectively (with a kappa coefficient of 0.8231), and the sensitivity of our revised LAMP assay was increased compared to existing methods. PMID:27109067

  18. MALARIA DIAGNOSIS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN THAILAND

    PubMed Central

    OCKER, Ronja; PROMPUNJAI, Yongyut; CHUTIPONGVIVATE, Salakchit; KARANIS, Panagiotis

    2016-01-01

    The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower. PMID:27074321

  19. Rapid detection of feline morbillivirus by a reverse transcription loop-mediated isothermal amplification

    PubMed Central

    KOIDE, Rie; SAKAGUCHI, Shoichi; OGAWA, Makoto; MIYAZAWA, Takayuki

    2015-01-01

    Feline morbillivirus (FmoPV) is an emerging virus in domestic cats and considered to be one of the causes of chronic renal failure in cats. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of FmoPV. The results indicated that the detection limit of the assay was 10 50% tissue culture infective dose (TCID50)/ml in the original sample, and sensitivity of the assay was calculated as 0.12 TCID50 per one RT-LAMP reaction. We also detected FmoPV in clinical urine samples from cats infected with FmoPV. The FmoPV RT-LAMP assay is rapid, simple and highly specific for the detection of FmoPV, and thus, it would be a reliable detection method for FmoPV. PMID:26268665

  20. Detection of red-spotted grouper nervous necrosis virus by loop-mediated isothermal amplification.

    PubMed

    Xu, Hai-Dong; Feng, Juan; Guo, Zhi-Xun; Ou, You-Jun; Wang, Jiang-Yong

    2010-01-01

    Red-spotted grouper nervous necrosis virus (RGNNV) causes high mortality in marine fish larvae cultured in China. To control better an outbreak of this virus, a rapid, specific and sensitive detection method based on loop-mediated isothermal amplification (LAMP) was developed. A set of four primers, two outer and two inner, was designed from RGNNV genome RNA. The LAMP reaction mix was optimized. The method was specific as no cross-reaction was observed between white spot syndrome virus, koi herpesvirus, infectious spleen and kidney necrosis virus, mud crab reovirus, and grass carp hemorrhage virus. The sensitivity of LAMP was 100-fold higher than the nested PCR in detecting the presence of RGNNV. RGNNV was detected in the brain of Trachinotus ovatus that showed typical symptoms of NNV infection, with the standardized LAMP procedure.

  1. [Microchip electrophoresis coupled with multiplex allele-specific am-plification for typing multiple single nucleotide polymorphisms (SNPs) simultaneously].

    PubMed

    Wang, Wei-Peng; Zhou, Guo-Hua

    2009-02-01

    A new method of DNA adapter ligation-mediated allele-specific amplification (ALM-ASA) was developed for typing multiple single nucleotide polymorphisms (SNPs) on the platform of microchip electrophoresis. Using seven SNPs of 794C>T, 1274C>T, 2143T>C, 2766T>del, 3298G>A, 5200G>A, and 5277C>T in the interleukin 1B (IL1B) gene as a target object, a long DNA fragment containing the seven SNPs of interest was pre-amplified to enhance the specificity. The pre-amplified DNA fragment was digested by a restriction endonuclease to form sticky ends; and then the adapter was ligated to either end of the digested fragment. Using the adapter-ligated fragments as templates, a 7-plex allele-specific amplification was performed by 7 allele-specific primers and a universal primer in one tube. The allele-specific products amplified were separated by chip electrophoresis and the types of SNPs were easily discriminated by the product sizes. The seven SNPs in IL1B gene in 48 healthy Chinese were successfully typed by microchip electrophoresis and the results coincided with those by PCR-restriction fragment length polymorphism and sequencing method. The method established was accurate and can be used to type multiple SNPs simultaneously. In combination with microchip electrophoresis for readout, ALM-ASA assay can be used for fast SNP detection with a small amount of sample. Using self-prepared gel matrix and reused chips for analysis, the SNP can be typed at an ultra low cost.

  2. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    PubMed

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples. PMID:25822163

  3. Detection of the food allergen celery via loop-mediated isothermal amplification technique.

    PubMed

    Zahradnik, Celine; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.

  4. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    PubMed

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

  5. Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification.

    PubMed

    Guo, Zhixun; Zhang, Di; Ma, Hongling; Su, Youlu; Feng, Juan; Xu, Liwen

    2014-11-01

    Mud crab dicistrovirus-1 (MCDV-1) was isolated from the mud crab (Scylla paramamosain), resulting in mass mortality and widespread economic loss in China. In this study, a detection method for MCDV-1 using loop-mediated isothermal amplification was developed. Two pairs of primers targeting the VP2 gene were designed. These primers were the outer primers F3 and B3, and the inner primers FIP and BIP. Optimal amplification was carried out using 0.2 μmol/L F3/B3, 1.6 μmol/L FIP/BIP, 6 mmol/L Mg(2+), 0.8 mmol/L dNTPs, and 0.8 mol/L betaine, and completed in 1h at 62°C. The products demonstrated a ladder pattern on agarose gel electrophoresis and could also be detected visually according to turbidity, or by adding SYBR Green I and observing a color change from orange to green. The proposed method could specifically amplify MCDV-1 gene fragments. Sensitivity assay revealed that six copies of the viral genome could be detected by this method, which was 1000-fold more sensitive than that of conventional PCR using constructed plasmid as amplification template. At clinical sample level, sensitivity of LAMP was 100-fold higher than that of conventional PCR.

  6. Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

  7. A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infections are common and contribute to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay with a bacterial enrichment proced...

  8. Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of P. infestans DNA. Methods and Results: Two sets of LAMP primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the ...

  9. Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay.

    PubMed

    Niessen, Ludwig; Vogel, Rudi F

    2010-06-15

    Loop-mediated isothermal amplification (LAMP) of DNA is a simple, cost effective, and rapid method for the specific detection of genomic DNA using a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from Geobacillus stearothermophilus for DNA amplification. The method has been applied in various assays for the diagnosis of bacterial and viral infections of humans and animals, sexing of bovine and swine embryos, and in the detection of bacteria from environmental samples. Only recently, first applications for fungal organisms were published. During the current study a LAMP assay was developed for the specific detection of Fusarium graminearum, the major causative agent of Fusarium head blight of small cereals and producer of the mycotoxins deoxynivalenol, nivalenol, and zearalenone. The assay was based on the gaoA gene (galactose oxidase) of the fungus. Amplification of DNA during the reaction was indirectly detected in situ by using calcein fluorescence as a marker without the necessity of time-consuming electrophoretic analysis. The assay was optimized for rapidness, specificity, and sensitivity and was shown to detect the presence of less than 2pg of purified target DNA per reaction within 30 min. Within 132 fungal species tested, exclusively DNA isolated from cultures of F. graminearum (lineages 1-9) resulted in a fluorescent signal after amplification with the LAMP assay. The method was demonstrated to be useful in the analysis of fungal cultures by direct analysis of surface scrapings from agar plate cultures, direct testing of single infected barley grains, and detection of F. graminearum in total genomic DNA isolated from bulk samples of ground wheat grains. Results obtained indicate that LAMP offers an interesting new assay format for the rapid and specific DNA-based detection and identification of agriculturally important toxigenic fungi in pure

  10. NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification.

    PubMed

    Borysiak, Mark D; Kimura, Kevin W; Posner, Jonathan D

    2015-04-01

    Nucleic acid amplification tests are the gold standard for many infectious disease diagnoses due to high sensitivity and specificity, rapid operation, and low limits of detection. Despite the advantages of nucleic acid amplification tests, they currently offer limited point-of-care (POC) utility due to the need for complex instruments and laborious sample preparation. We report the development of the Nucleic Acid Isotachophoresis LAMP (NAIL) diagnostic device. NAIL uses isotachophoresis (ITP) and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. ITP is an electrokinetic separation technique that uses an electric field and two buffers to extract and purify nucleic acids in a single step. LAMP amplifies nucleic acids at constant temperature and produces large amounts of DNA that can be easily detected. A mobile phone images the amplification results to eliminate the need for laser fluorescent detection. The device requires minimal user intervention because capillary valves and heated air chambers act as passive valves and pumps for automated fluid actuation. In this paper, we describe NAIL device design and operation, and demonstrate the extraction and detection of pathogenic E. coli O157:H7 cells from whole milk samples. We use the Clinical and Laboratory Standards Institute (CLSI) limit of detection (LoD) definitions that take into account the variance from both positive and negative samples to determine the diagnostic LoD. According to the CLSI definition, the NAIL device has a limit of detection (LoD) of 1000 CFU mL(-1) for E. coli cells artificially inoculated into whole milk, which is two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples and comparable to lab-based methods. The NAIL device potentially offers significant reductions in the complexity and cost of traditional nucleic acid diagnostics for POC applications

  11. NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification.

    PubMed

    Borysiak, Mark D; Kimura, Kevin W; Posner, Jonathan D

    2015-04-01

    Nucleic acid amplification tests are the gold standard for many infectious disease diagnoses due to high sensitivity and specificity, rapid operation, and low limits of detection. Despite the advantages of nucleic acid amplification tests, they currently offer limited point-of-care (POC) utility due to the need for complex instruments and laborious sample preparation. We report the development of the Nucleic Acid Isotachophoresis LAMP (NAIL) diagnostic device. NAIL uses isotachophoresis (ITP) and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. ITP is an electrokinetic separation technique that uses an electric field and two buffers to extract and purify nucleic acids in a single step. LAMP amplifies nucleic acids at constant temperature and produces large amounts of DNA that can be easily detected. A mobile phone images the amplification results to eliminate the need for laser fluorescent detection. The device requires minimal user intervention because capillary valves and heated air chambers act as passive valves and pumps for automated fluid actuation. In this paper, we describe NAIL device design and operation, and demonstrate the extraction and detection of pathogenic E. coli O157:H7 cells from whole milk samples. We use the Clinical and Laboratory Standards Institute (CLSI) limit of detection (LoD) definitions that take into account the variance from both positive and negative samples to determine the diagnostic LoD. According to the CLSI definition, the NAIL device has a limit of detection (LoD) of 1000 CFU mL(-1) for E. coli cells artificially inoculated into whole milk, which is two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples and comparable to lab-based methods. The NAIL device potentially offers significant reductions in the complexity and cost of traditional nucleic acid diagnostics for POC applications.

  12. Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

    PubMed Central

    Kubota, Ryo; Jenkins, Daniel M.

    2015-01-01

    Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field. PMID:25741765

  13. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

    PubMed

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane. PMID:27014303

  14. Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs.

    PubMed

    Mandal, Mrityunjay; Banerjee, Partha S; Kumar, Saroj; Ram, Hira; Garg, Rajat; Pawde, Abhijit M

    2015-04-15

    Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting. PMID:25749021

  15. Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis.

    PubMed

    Yang, Wang; Ying, Fang; Yingyu, Liu; Pin, Chen; Wentao, Li; Shuqing, Liu; Haoyong, Zou; Qigai, He

    2010-12-01

    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.

  16. Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae.

    PubMed

    Yang, Wang; Pin, Chen; Haibing, Guo; Yang, Chen; Hui, Liu; Qigai, He

    2009-11-01

    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (chi(2) test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.

  17. Real-time loop-mediated isothermal DNA amplification in compact disc micro-reactors.

    PubMed

    Santiago-Felipe, Sara; Tortajada-Genaro, Luis A; Carrascosa, Javier; Puchades, Rosa; Maquieira, Ángel

    2016-05-15

    An integrated device composed of micro-reactors embedded onto compact discs is proposed for real-time targeted DNA determination. The method principle is based on in-disc loop-mediated isothermal amplification (iD-LAMP) and quantitative optical read-out by a disc drive. In the presence of a target, the turbidimetric or colorimetric properties of reaction solution change, and the transmitted intensity of the disc drive laser modifies according to reaction yield. Monitoring real-time curves allowed the quantitative determination of DNA template amounts. The best amplification/detection results were obtained with micro-reactors (2mm diameter and 1.1mm in depth) drilled on a digital video disc (DVD) and detection based on the colorimetric mode. As proof-of-concept, the assay was applied to detect pathogenic bacteria Salmonella spp. and to identify bovine meat in food samples. Ninety-six samples were simultaneously analysed in 15 min, with high selectivity and sensitivity (5 CFU/mL and 10 µg/g for bacteria and meat, respectively). The in-disc results were comparable to those obtained by conventional LAMP or qPCR approaches. The developed device allows low sample and reagent consumption (3 µL of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses. PMID:26716424

  18. Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola

    PubMed Central

    Kong, Xiangjiu; Qin, Wentao; Huang, Xiaoqing; Kong, Fanfang; Schoen, Cor D.; Feng, Jie; Wang, Zhongyue; Zhang, Hao

    2016-01-01

    A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-μL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estimating the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extraction method, this assay’s total detection time is shortened to approximately one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly associated with disease severity. The LAMP method was also demonstrated to be able to estimate the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew. PMID:27363943

  19. Detection of mink enteritis virus by loop-mediated isothermal amplification (LAMP).

    PubMed

    Wang, Jianke; Cheng, Shipeng; Yi, Li; Cheng, Yuening; Yang, Shen; Xu, Hongli; Li, Zhenguang; Shi, Xinchuan; Wu, Hua; Yan, Xijun

    2013-02-01

    Loop-mediated isothermal amplification (LAMP) method was discovered in the last decade but only used for the first time in the diagnosis of mink enteritis virus (MEV) infection in this study. The amplification could be completed within 60 min, under isothermal condition at 65°C, by employing a set of four primers targeting the VP2 gene of MEV. The LAMP was more sensitive than the conventional PCR, with a detection limit of 10(-1) median tissue culture infective doses (TCID(50))/ml per reaction, compared with 10 TCID(50)/ml for PCR analysis. No cross reactivity was observed for other related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). Eighty four of 230 clinical samples were found to be positive for MEV, which is higher than that determined by using the conventional PCR method (68). The results indicate the LAMP can be potentially used to determine MEV as a simple, rapid procedure. This assay would be an available alternative to PCR analysis for the diagnosis of MEV infection in mink, particularly in less well-equipped laboratories and in rural settings where resources are limited. PMID:23183142

  20. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

  1. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane. PMID:27014303

  2. The use of loop-mediated isothermal amplification improves Toxoplasma gondii detection in wildlife.

    PubMed

    Trisciuoglio, Anna; Zanet, Stefania; Marello, Giuseppina; Chiesa, Francesco; Nucera, Daniele M; Bergallo, Massimiliano; Gennero, Maria S; Ferroglio, Ezio

    2015-11-01

    Toxoplasma gondii is among the most widespread parasites worldwide. Wildlife is recognized as an important reservoir and source of infection of T. gondii. The goal of the present work was to assess the performance of loop-mediated isothermal amplification (LAMP) as a diagnostic tool for T. gondii infection in the skeletal muscle and central nervous system (CNS) of free-ranging ungulates and carnivores. Fifty-seven wild animals were tested for the presence of T. gondii DNA by polymerase chain reaction (PCR) and LAMP. The use of LAMP amplification improved sensitivity in T. gondii molecular detection compared with conventional PCR on skeletal muscle (χ(2) = 5.8, P < 0.05), having a lower minimum detection limit (0.1 tachyzoite) than PCR (1 tachyzoite). No significant differences existed between the detection capacities of both assays when performed on CNS. LAMP is a valid tool to improve the diagnosis of T. gondii infection in wild game meat. The technique provides a sensitive yet specific method that can be applicable to both field surveys and large-scale testing of wildlife samples. PMID:26462761

  3. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

    PubMed

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane.

  4. Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis.

    PubMed

    Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

    2016-06-01

    Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications. PMID:27417089

  5. Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry.

    PubMed

    Tian, Bo; Qiu, Zhen; Ma, Jing; Zardán Gómez de la Torre, Teresa; Johansson, Christer; Svedlindh, Peter; Strömberg, Mattias

    2016-12-15

    Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control. PMID:27423039

  6. Rapid detection of European orthobunyaviruses by reverse transcription loop-mediated isothermal amplification assays.

    PubMed

    Camp, Jeremy V; Nowotny, Norbert

    2016-10-01

    The development of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assays are described herein for the detection of two orthobunyaviruses (Bunyaviridae), which represent the two main serogroups found in mosquitoes in Central Europe. The RT-LAMP assays were optimized for the detection of Ťahyňa virus (a California encephalitis group virus found in Aedes sp or Ochlerotatus sp mosquitoes) and Batai virus (also called Čalovo virus, a Bunyamwera group virus found in Anopheles maculipennis s.l. mosquitoes) nucleic acid using endemic European virus isolates. The sensitivity of the RT-LAMP assays was determined to be comparable to that of conventional tests, with a limit of detection<0.1 pfu per reaction. The assays can be performed in 60min under isothermal conditions using very simple equipment. Furthermore, it was possible to proceed with the assays without nucleic acid extraction, albeit at a 100-fold loss of sensitivity. The RT-LAMP assays are a sensitive, cost-efficient method for both arbovirus surveillance as well as diagnostic laboratories to detect the presence of these endemic orthobunyaviruses.

  7. Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification.

    PubMed

    Wang, Xin-Ru; Wu, Li-Fen; Wang, Yan; Ma, Ying-Ying; Chen, Feng-Hua; Ou, Hong-Ling

    2015-01-01

    Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 °C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg/μl DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg/μl and 147 pg/μl DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity.

  8. Rapid detection of European orthobunyaviruses by reverse transcription loop-mediated isothermal amplification assays.

    PubMed

    Camp, Jeremy V; Nowotny, Norbert

    2016-10-01

    The development of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assays are described herein for the detection of two orthobunyaviruses (Bunyaviridae), which represent the two main serogroups found in mosquitoes in Central Europe. The RT-LAMP assays were optimized for the detection of Ťahyňa virus (a California encephalitis group virus found in Aedes sp or Ochlerotatus sp mosquitoes) and Batai virus (also called Čalovo virus, a Bunyamwera group virus found in Anopheles maculipennis s.l. mosquitoes) nucleic acid using endemic European virus isolates. The sensitivity of the RT-LAMP assays was determined to be comparable to that of conventional tests, with a limit of detection<0.1 pfu per reaction. The assays can be performed in 60min under isothermal conditions using very simple equipment. Furthermore, it was possible to proceed with the assays without nucleic acid extraction, albeit at a 100-fold loss of sensitivity. The RT-LAMP assays are a sensitive, cost-efficient method for both arbovirus surveillance as well as diagnostic laboratories to detect the presence of these endemic orthobunyaviruses. PMID:27491341

  9. Detection of the Quarantine Species Thrips palmi by Loop-Mediated Isothermal Amplification

    PubMed Central

    Przybylska, Arnika; Fiedler, Żaneta; Kucharczyk, Halina; Obrępalska-Stęplowska, Aleksandra

    2015-01-01

    Thrips palmi (from the order Thysanoptera) is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union) as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template, indicating that the DNA isolation step could be omitted. The described method is species-specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field. PMID:25793743

  10. Detection of Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification.

    PubMed

    Yasuhara-Bell, Jarred; de Silva, Asoka; Heuchelin, Scott A; Chaky, Jennifer L; Alvarez, Anne M

    2016-03-01

    The Goss's wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss's wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms. PMID:26595113

  11. Development of Lyophilized Loop-Mediated Isothermal Amplification Reagents for the Detection of Leptospira.

    PubMed

    Chen, Hua-Wei; Weissenberger, Giulia; Ching, Wei-Mei

    2016-05-01

    Leptospirosis is considered to be the most widespread zoonosis. This worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus Leptospira. Polymerase chain reaction (PCR)-based diagnostic assays have been developed for detecting Leptospira DNA in cell cultures and clinical samples. Because PCR requires specialized equipment and extensive end-user training, it is not suitable for routine work in resource-limited areas. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of Leptospira in patient, animal and environmental samples using lyophilized reagents at a single temperature of around 63°C with a heating block. The sensitivity of this LAMP assay is very similar to the PCR method. The amplified DNA products can be visualized with the naked eyes using hydroxy naphthol blue or detected by the fluorescence signal of SYBR green dye in the reaction when an ultraviolet lamp or compact fluorescence tube scanner is used. This LAMP assay is simple, rapid, and can be performed with a water bath or heating block. The lyophilized LAMP reagents are stable for 3 months when stored at 4°C and 1 month when stored at 25°C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic. PMID:27168577

  12. The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii.

    PubMed

    Chen, Hua-Wei; Ching, Wei-Mei

    2016-04-18

    Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 °C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

  13. Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

    NASA Astrophysics Data System (ADS)

    Gao, Hongwei; Li, Fuhua; Zhang, Xiaojun; Wang, Bing; Xiang, Jianhai

    2010-01-01

    Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase ( empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non- V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.

  14. Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis.

    PubMed

    Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

    2016-06-01

    Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.

  15. Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis

    PubMed Central

    Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

    2016-01-01

    Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications. PMID:27417089

  16. A Reverse Transcription Loop-Mediated Isothermal Amplification Assay Optimized to Detect Multiple HIV Subtypes

    PubMed Central

    Ocwieja, Karen E.; Sherrill-Mix, Scott; Liu, Changchun; Song, Jinzhao; Bau, Haim; Bushman, Frederic D.

    2015-01-01

    Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26), targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable). There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1) tracking response to medication by comparing longitudinal values for a subject, 2) detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3) detecting infection prior to seroconversion. PMID:25675344

  17. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

    PubMed

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2016-07-01

    In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control. PMID:27118244

  18. Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira.

    PubMed

    Chen, Hua-Wei; Weissenberger, Giulia; Atkins, Erin; Chao, Chien-Chung; Suputtamongkol, Yupin; Ching, Wei-Mei

    2015-01-01

    Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

  19. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

    PubMed

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2016-07-01

    In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control.

  20. Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection.

    PubMed

    Oh, Seung Jun; Park, Byung Hyun; Jung, Jae Hwan; Choi, Goro; Lee, Doh C; Kim, Do Hyun; Seo, Tae Seok

    2016-01-15

    We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection.

  1. A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

    PubMed

    Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

    2014-12-01

    Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative. PMID:25776596

  2. Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry.

    PubMed

    Tian, Bo; Qiu, Zhen; Ma, Jing; Zardán Gómez de la Torre, Teresa; Johansson, Christer; Svedlindh, Peter; Strömberg, Mattias

    2016-12-15

    Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

  3. Endpoint Visual Detection of Three Genetically Modified Rice Events by Loop-Mediated Isothermal Amplification

    PubMed Central

    Chen, Xiaoyun; Wang, Xiaofu; Jin, Nuo; Zhou, Yu; Huang, Sainan; Miao, Qingmei; Zhu, Qing; Xu, Junfeng

    2012-01-01

    Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%–0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops. PMID:23203072

  4. The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii.

    PubMed

    Chen, Hua-Wei; Ching, Wei-Mei

    2016-01-01

    Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 °C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic. PMID:27167168

  5. Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus

    PubMed Central

    Batra, Kanisht; Kumar, Aman; Kumar, Vinay; Nanda, Trilok; Maan, Narender S; Maan, Sushila

    2015-01-01

    Aim: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR). Material and Methods: Lyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. Results: The test described is quick (30 min), sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR). LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. Conclusion: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments. PMID:27047031

  6. Reverse transcription loop-mediated isothermal amplification assay for detecting tomato chlorosis virus.

    PubMed

    Zhao, Li-ming; Li, Gang; Gao, Ying; Zhu, You-rong; Liu, Jin; Zhu, Xiao-ping

    2015-03-01

    A betaine-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimised for detecting tomato chlorosis virus (ToCV), one of the most important viruses that infect tomato crops worldwide. A set of four specific primers was designed against the RNA-dependent RNA polymerase (RdRp) gene. The betaine-free RT-LAMP procedure could be completed within 40 min under isothermal conditions at 60 °C without a thermal cycler, and no cross-reactivity was seen with other tomato viral pathogens. Sensitivity analysis showed that RT-LAMP could detect viral dilutions up to 2.0×10(-7)ng, which is 100-times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR). In addition, naked-eye observation after staining in-tube RT-LAMP products with SYBR Green I facilitated detection of ToCV by avoiding the requirement for ethidium staining following gel electrophoresis. These results suggest that ToCV RT-LAMP is a rapid, sensitive, and affordable diagnostic tool that is more suitable than RT-PCR for the detection and surveillance of ToCV in field samples. PMID:25486081

  7. Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification.

    PubMed

    Kuan, Cheng-Ping; Wu, Min-Tze; Lu, Yi-Lin; Huang, Hung-Chang

    2010-10-01

    A loop-mediated isothermal amplification (LAMP) assay was employed to develop a simple and efficient system for the detection of squash leaf curl virus (SLCV) in diseased plants of squash (Cucurbita pepo) and melon (Cucumis melo). Completion of LAMP assay required 30-60 min under isothermal conditions at 65 degrees C by employing a set of four primers targeting SLCV. Although the sensitivity of the LAMP assay and the polymerase chain reaction (PCR) assay was comparable at high virus concentrations, the LAMP assay was by a 10-fold dilution factor more sensitive than the PCR assay for the detection of SLCV in diseased plants. No reaction was detected in the tissues of healthy plants by either the LAMP or the PCR. The LAMP products can be visualized by staining directly in the tube with SYBR Safe DNA gel stain dye. The sensitivity of the SYBR Safe DNA gel stain is similar to analysis by gel electrophoresis. Although both the LAMP and the PCR methods were capable of detecting SLCV in infected tissues of squash and melon, the LAMP method would be more useful than the PCR method for detection of SLCV infection in cucurbitaceous plants because it is more rapid, simple, accurate and sensitive.

  8. Molecular detection of Coxiella burnetii using an alternative loop-mediated isothermal amplification assay (LAMP).

    PubMed

    Raele, Donato Antonio; Garofolo, Giuliano; Galante, Domenico; Cafiero, Maria Assunta

    2015-01-01

    Q fever, caused by Coxiella burnetii, is a worldwide zoonosis with important consequences for human and animal health. In livestock, the diagnosis, using direct and indirect techniques, is challenging even if to tackle coxiellosis in domesticated animals a rapid diagnosis is crucial. In the recent years, new molecular methods have been developed to overcome these issues. Several polymerase chain reaction (PCR) assays have been studied, but loop mediated isothermal amplification (LAMP) has not been fully developed. This new methodology is emerging due to simplicity and speed in diagnosis of microbial diseases. In this study, we design a new LAMP assay against C. burnetii targeting the com1 gene as an actual alternative to conventional PCR. The assay was specific to C. burnetii reactive with sensitivity comparable to standard PCR. The application of the com1 LAMP on 10 clinical samples from water buffalo, sheep, and goats, previously tested positive, confirmed the presence of C. burnetii. To our knowledge, this study is the first report of LAMP targeting C. burnetii in Europe and the results also suggest that it may be an useful and cost-effective tool for the clinical and epidemiological surveillance of Q Fever.

  9. Simple detection of Pythium irregulare using loop-mediated isothermal amplification assay.

    PubMed

    Feng, Wenzhuo; Ishiguro, Yasushi; Hotta, Keisuke; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2015-11-01

    Pythium irregulare is an important soil-borne pathogen that causes seed, stem and root rot, and seedling damping-off in various crops. Here, we have developed a rapid and reliable approach for detecting the pathogen using loop-mediated isothermal amplification (LAMP) in combination with primers designed from the sequences of the P. irregulare ribosomal DNA internal transcribed spacer region. The specificity of the primers for P. irregulare was tested using 50 isolates of 40 Pythium species, 11 Phytophthora isolates and 8 isolates of 7 other soil-borne pathogens. The assay showed that the limit of sensitivity of the LAMP method was 100 fg of pure DNA, a similar level to that of a polymerase chain reaction. LAMP detected P. irregulare from the supernatant after mixing culture medium (template DNA source) with distilled water. Similarly, positive results were obtained using a 'Plant-LAMP' method applied to a suspension rotted roots in water. A 'Bait-LAMP' method using the supernatant of autoclaved perilla seeds incubated in a soil/water mixture for 1 week at 25°C successfully detected P. irregulare from the soil. The LAMP assay described in this study is therefore a simple and effective way for practical detection of P. irregulare.

  10. Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

    PubMed Central

    2014-01-01

    Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07℃ for cattle, 84.96±0.08℃ for pig, and 85.99±0.05℃ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11℃ for goat and 83.90±0.11℃ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23℃ for chicken, 88.66±0.12℃ for duck, and 84.49±0.08℃ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/μL to 100 fg/μL levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species. PMID:26761677

  11. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay.

    PubMed

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL(-1) of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics. PMID:27625648

  12. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics. PMID:27625648

  13. A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

    PubMed Central

    Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

    2015-01-01

    Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation

  14. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology.

    PubMed

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10(-3) for bcr1 and bcr3 and 10(-)2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL. PMID:25815362

  15. Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.

    PubMed

    Khamlor, Trisadee; Pongpiachan, Petai; Parnpai, Rangsun; Punyawai, Kanchana; Sangsritavong, Siwat; Chokesajjawatee, Nipa

    2015-03-15

    In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing. PMID:25542460

  16. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

    PubMed Central

    Lucchi, Naomi W.; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  17. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    PubMed

    Lucchi, Naomi W; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

  18. Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification

    PubMed Central

    Ranjbar, Reza; Erfanmanesh, Maryam; Afshar, Davoud; Mohammadi, Mohsen; Ghaderi, Omar; Haghnazari, Ali

    2016-01-01

    Introduction Escherichia coli O157:H7, an important foodborne pathogen, can cause serious renal damage, which can also lead to mortality. Since a rapid and sensitive method is needed to identify this pathogenic agent, we evaluated Loop-Mediated Isothermal Amplification Assay (LAMP) to detect Escherichia coli O157:H7. Methods We used six primers that specifically identified the rfbE gene. To examine the sensitivity of the method, different dilutions were subjected to the LAMP reaction. Other bacterial strains also were investigated to determine the specificity of the test. The turbidity of the amplified products was assayed by visual detection. The amplified products were detected by addition of SYBR Green II to the reaction tubes. Results Amplification products were observed as a ladder-like pattern on the agarose gel. A white turbidity emerged in the positive tubes. Under UV light, the positive samples were green, whereas the negative samples were orange. The detection limit of the LAMP was 78 pg/tube, and this indicated that it was 100 times more sensitive than PCR for the detection of EHEC. No LAMP products were detected when template DNA of non-EHEC strains were used, suggesting high specificity of the LAMP assay. Conclusion The results indicated that the LAMP assay is a valuable diagnostic assay to identify EHEC O157:H7. In addition, the simplicity, sensitivity, specificity, and rapidity of this assay make it a useful method to diagnose pathogens in primary labs without any need for expensive equipment or specialized techniques. PMID:27504175

  19. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    PubMed

    Lucchi, Naomi W; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  20. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology

    PubMed Central

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10−3 for bcr1 and bcr3 and 10−2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL. PMID:25815362

  1. Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification.

    PubMed

    Hirayama, Hiroki; Kageyama, Soichi; Takahashi, Yoshiyuki; Moriyasu, Satoru; Sawai, Ken; Onoe, Sadao; Watanabe, Keiko; Kojiya, Shinichi; Notomi, Tsugunori; Minamihashi, Akira

    2006-09-15

    Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.

  2. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    SciTech Connect

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  3. Loop-mediated isothermal amplification for rapid and semiquantitative detection of Loa loa infection.

    PubMed

    Drame, Papa M; Fink, Doran L; Kamgno, Joseph; Herrick, Jesica A; Nutman, Thomas B

    2014-06-01

    Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic. PMID:24696020

  4. A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus.

    PubMed

    Min, Zhang; Hongli, Jing; Lifeng, Zhang; Na, Wang; Shaoqiang, Wu; Xiangmei, Lin

    2013-10-01

    A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 μL of each inner primer, RV-FIP (20 pmol/μL) and RV-BIP (20 pmol/μL), 0.5 μL of each outer primer, RV-F3 (10 pmol/μL) and RV-B3 (10 pmol/μL), 1.25 μL of each loop primer, RV-LF (20 pmol/μL) and RV-LB (20 pmol/μL), 3.5 μL dNTP mix (10 mM each), 8 μL MgSO4 (25 mM), 1 μL of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 μL 10 × supplied buffer, and 1 μL of template DNA in a final volume of 25 μL. The optimum reaction conditions were 63 °C for 60 min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added.

  5. Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses.

    PubMed

    Das, Amaresh; Babiuk, Shawn; McIntosh, Michael T

    2012-05-01

    Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited.

  6. Loop-Mediated Isothermal Amplification for Rapid and Semiquantitative Detection of Loa loa Infection

    PubMed Central

    Drame, Papa M.; Fink, Doran L.; Kamgno, Joseph; Herrick, Jesica A.

    2014-01-01

    Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic. PMID:24696020

  7. Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP).

    PubMed

    Obura, E; Masiga, D; Wachira, F; Gurja, B; Khan, Z R

    2011-02-01

    Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas. PMID:21185882

  8. Visual detection of goose haemorrhagic polyomavirus in geese and ducks by loop-mediated isothermal amplification.

    PubMed

    Woźniakowski, Grzegorz; Tarasiuk, Karolina

    2015-01-01

    Goose haemorrhagic polyomavirus (GHPV) is an aetiological agent of haemorrhagic nephritis and enteritis of geese occurring in geese (Anser anser). GHPV may also infect Muscovy ducks (Carina mochata) and mule ducks. Early detection of GHPV is important to isolate the infected birds from the rest of the flock thus limiting infection transmission. The current diagnosis of haemorrhagic nephritis and enteritis of geese is based on virus isolation, histopathological examination, haemagglutination inhibition assay, ELISA and polymerase chain reaction (PCR). Recently, real-time PCR assay was developed which considerably improved detection of GHPV. In spite of many advantages, these methods are still time-consuming and inaccessible for laboratories with limited access to ELISA plate readers or PCR thermocyclers. The aim of our study was to develop loop-mediated isothermal amplification (LAMP) that may be conducted in a water bath. Two pairs of specific primers complementary to VP1 gene of GHPV were designed. The results of GHPV LAMP were recorded under ultraviolet light. Our study showed LAMP was able to specifically amplify VP1 fragment of a GHPV without cross-reactivity with other pathogens of geese and ducks. LAMP detected as little as 1.5 pg of DNA extracted from a GHPV standard strain (150 pg/µl). The optimized LAMP was used to examine 18 field specimens collected from dead and clinically diseased geese and ducks aged from 1 to 12 weeks. The positive signal for GHPV was detected in three out of 18 (16.6%) specimens. These results were reproducible and consistent with those of four real-time PCR. To the best of our knowledge this is the first report on LAMP application for the GHPV detection.

  9. Development of primer sets for loop-mediated isothermal amplification that enables rapid and specific detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three ...

  10. Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping.

    PubMed

    Wu, Wei-Ming; Tsai, Hsiang-Ju; Pang, Jong-Hwei S; Wang, Tzu-Hao; Wang, Hsin-Shih; Hong, Hong-Shang; Lee, Yun-Shien

    2005-10-01

    Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.

  11. [Advance in loop-mediated isothermal amplification technique and its applications in point-of-care testing platforms].

    PubMed

    Guan, Li; Ma, Xue-Jun

    2014-07-01

    Loop-mediated isothermal amplification (LAMP) is a novel in vitro nucleic acid amplification method conducted under isothermal conditions with the advantages of high specificity, sensitivity, rapidity and easy detection. Since it was established in 2000, it has been widely applied in various fields of analytical science including the diagnosis of a variety of pathogens, identification of embryo sex, detection of genetically modified organisms and cancer gene identification. Additionally, significant progress has been made in the optimization of the LAMP method, such as accelerated reactions, simplified sample processing, the realization of multiplex amplification, and the enhanced specificity of reaction and detection methods. LAMP technology also shows much potential to be adopted as part of point-of-care testing platforms by the micromation, automation and integration with other technologies such as Lab-on-a-Chip and digital nucleic acid amplification. This review summarizes the latest advances in the LAMP technique and its applications in developing point-of-care testing platforms.

  12. The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA.

    PubMed

    Billard, A; Laval, V; Fillinger, S; Leroux, P; Lachaise, H; Beffa, R; Debieu, D

    2012-02-01

    The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

  13. Development of loop-mediated isothermal amplification for detection of Leifsonia xyli subsp. xyli in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is prevalent in most sugarcane-planting countries. Because the disease does not cause characteristic external symptoms, a laboratory-based technique is needed for accurate diagnosis. Based on loop-mediat...

  14. Loop-mediated isothermal amplification (LAMP) based detection of Colletotrichum falcatum causing red rot in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red rot, caused by Colletotrichum falcatum, is a destructive disease prevalent in most sugarcane-producing countries. Disease-free sugarcane planting materials are essential as the pathogen spreads primarily through infected setts. The present study was undertaken to develop loop-mediated isothermal...

  15. Using the ubiquitous pH meter combined with a loop mediated isothermal amplification method for facile and sensitive detection of Nosema bombycis genomic DNA PTP1.

    PubMed

    Xie, Shunbi; Yuan, Yali; Song, Yue; Zhuo, Ying; Li, Tian; Chai, Yaqin; Yuan, Ruo

    2014-12-28

    Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during the loop mediated isothermal amplification (LAMP) procedure by using a pH meter. The genomic DNA of Nosema bombycis (N. bombycis) was amplified and detected by employing this LAMP-pH meter platform for the first time.

  16. Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus.

    PubMed

    Oyhenart, Jorge; Martínez, Florencia; Ramírez, Rosana; Fort, Marcelo; Breccia, Javier D

    2013-03-31

    Tritrichomonas foetus is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to infertility and abortion. A test based on loop mediated isothermal amplification (LAMP) targeting the 5.8S rDNA subunit was designed for the specific identification of T. foetus. The LAMP assay was validated using 28 T. foetus and 35 non-T. foetus trichomonads strains. It did not exhibit cross-reaction with closely related parasites commonly found in smegma cultures like Tetratrichomonas spp. and Pentatrichomonas hominis. Bovine smegma did not show interferences for the detection of the parasite and, the sensitivity of the method (4×10(3) CFU/mL, approximately 10 cells/reaction) was slightly higher than that found for PCR amplification with TFR3 and TFR4 primers. The LAMP approach has potential applications for diagnosis and control of T. foetus and, practical use for low skill operators in rural areas.

  17. Detection of cucumber mosaic virus isolates from banana by one-step reverse transcription loop-mediated isothermal amplification.

    PubMed

    Peng, Jun; Shi, Minjing; Xia, Zihao; Huang, Junsheng; Fan, Zaifeng

    2012-11-01

    Cucumber mosaic virus (CMV) is one of the most devastating threats to the banana industry. A single-tube, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of CMV-infected banana and plantain (Musa spp.). The reaction was performed in a single tube at 63 °C for 90 min using a real-time turbidimeter, with an improved closed-tube visual detection system in which fluorescent dye was added to the inside of the lid prior to amplification. This RT-LAMP assay is an alternative method for the rapid detection of CMV in banana plants and tissue culture materials. PMID:22782136

  18. Detection of cucumber mosaic virus isolates from banana by one-step reverse transcription loop-mediated isothermal amplification.

    PubMed

    Peng, Jun; Shi, Minjing; Xia, Zihao; Huang, Junsheng; Fan, Zaifeng

    2012-11-01

    Cucumber mosaic virus (CMV) is one of the most devastating threats to the banana industry. A single-tube, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of CMV-infected banana and plantain (Musa spp.). The reaction was performed in a single tube at 63 °C for 90 min using a real-time turbidimeter, with an improved closed-tube visual detection system in which fluorescent dye was added to the inside of the lid prior to amplification. This RT-LAMP assay is an alternative method for the rapid detection of CMV in banana plants and tissue culture materials.

  19. Protein detection through different platforms of immuno-loop-mediated isothermal amplification

    PubMed Central

    2013-01-01

    Different immunoassay-based methods have been devised to detect protein targets. These methods have some challenges that make them inefficient for assaying ultra-low-amounted proteins. ELISA, iPCR, iRCA, and iNASBA are the common immunoassay-based methods of protein detection, each of which has specific and common technical challenges making it necessary to introduce a novel method in order to avoid their problems for detection of target proteins. Here we propose a new method nominated as ‘immuno-loop-mediated isothermal amplification’ or ‘iLAMP’. This new method is free from the problems of the previous methods and has significant advantages over them. In this paper we also offer various configurations in order to improve the applicability of this method in real-world sample analyses. Important potential applications of this method are stated as well. PMID:24237767

  20. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Babesia canis infections.

    PubMed

    Müller, H; Aysul, N; Liu, Z; Salih, D A; Karagenc, T; Beyer, D; Kullmann, B; Ahmed, J S; Seitzer, U

    2010-04-01

    Vector-borne diseases are rising in interest due to global warming, which is believed to impact on the distribution of vectors into new areas thus influencing the occurrence and epidemiology of vector-borne pathogens. Babesia canis belongs to the Piroplasmidae and there are three described subspecies, namely B. canis canis, B. canis rossi and B. canis vogeli. They are each transmitted by a different tick-species, Dermacentor reticulatus, Haemaphysalis leachi and Rhipicephalus sanguineus, respectively. There are also differences in the geographical distribution and pathogenicity to dogs of each subspecies. In this study, we aimed to establish a rapid and easy to perform DNA-based test using loop-mediated isothermal amplification to detect all three Babesia canis subspecies in one assay.

  1. Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

    PubMed

    Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

    2015-01-01

    Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control. PMID:25584616

  2. Loop-Mediated Isothermal Amplification (LAMP) for Detection of Campylobacter jejuni and C. coli in Thai Children with Diarrhea.

    PubMed

    Pham, Ngan Thi Kim; Trinh, Quang Duy; Khamrin, Pattara; Ukarapol, Nuthapong; Kongsricharoern, Tipachan; Yamazaki, Wataru; Komine-Aizawa, Shihoko; Okitsu, Shoko; Maneekarn, Niwat; Hayakawa, Satoshi; Ushijima, Hiroshi

    2015-01-01

    Campylobacter species are common causes of bacterial diarrhea, and Campylobacter jejuni and C. coli are known as the predominant causative agents in humans. Recent studies suggested that loop-mediated isothermal amplification (LAMP) is an efficient and practical tool for rapid detection of C. jejuni and C. coli in clinical samples. We used LAMP to screen 151 stool samples for Campylobacter; these samples were collected in 2012 from Thai children with diarrhea. The PCR method discriminated C. jejuni and C. coli among the detected Campylobacter strains; these species were subjected to sequencing of the hipO gene (in C. jejuni) or the ask gene (in C. coli). The results suggest that the prevalence of Campylobacter infection among Thai children with diarrhea is 8.6%, and C. jejuni is the most prevalent species.

  3. Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

    PubMed

    Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

    2013-11-01

    In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

  4. Development of loop-mediated isothermal amplification method for detecting Wuchereria bancrofti DNA in human blood and vector mosquitoes.

    PubMed

    Takagi, Hidekazu; Itoh, Makoto; Kasai, Shinji; Yahathugoda, Thishan C; Weerasooriya, Mirani V; Kimura, Eisaku

    2011-12-01

    We have developed loop-mediated isothermal amplification (LAMP) method to detect Wuchereria bancrofti DNA. The sensitivity and specificity of LAMP method were equivalent to those of PCR method which detects SspI repeat sequence in W. bancrofti genomic DNA: both methods detected one thousandth of W. bancrofti DNA from one microfilaria (Mf), and did not cross-react with DNAs of Brugia malayi, B. pahangi, Dirofilaria immitis, human and Culex quinquefasciatus. We also examined the sensitivity of LAMP using the mimic samples of patient's blood or blood-fed mosquitoes containing one W. bancrofti Mf per sample. The LAMP method was able to detect W. bancrofti DNA in 1000 μl of blood or in a pool of 60 mosquitoes, indicating its usefulness in detecting/monitoring W. bancrofti infection in humans and vector mosquitoes in endemic areas. PMID:21930238

  5. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  6. Determination of ABO blood group genotypes using the real-time loop-mediated isothermal amplification method

    PubMed Central

    ZHANG, CHAO; ZHU, JUANLI; YANG, JIANGCUN; WAN, YINSHENG; MA, TING; CUI, YALI

    2015-01-01

    ABO genotyping is commonly used in several situations, including blood transfusion, personal identification and disease detection. The present study developed a novel method for ABO genotyping, using loop-mediated isothermal amplification (LAMP). This method allows the simultaneous determination of six ABO genotypes under 40 min at a constant temperature of 62°C. The genotypes of 101 blood samples were determined to be AA (n=6), AO (n=38), BB (n=12), BO (n =29), AB (n=8) and OO (n=8) by the LAMP assay. The results were compared with the phenotypes determined by serological assay and the genotypes determined by direct sequencing, and no discrepancies were observed. This novel and rapid method, with good accuracy and reasonably cost effective, provides a supplement to routine serological ABO typing and may also be useful in other point-of-care testing. PMID:26238310

  7. Rapid and Sensitive Identification of the Herbal Tea Ingredient Taraxacum formosanum Using Loop-Mediated Isothermal Amplification

    PubMed Central

    Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

    2015-01-01

    Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control. PMID:25584616

  8. Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

    PubMed

    Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

    2015-01-09

    Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

  9. A reverse transcription loop-mediated isothermal amplification method for rapid detection of bovine viral diarrhea virus.

    PubMed

    Fan, Qing; Xie, Zhixun; Xie, Liji; Liu, Jiabo; Pang, Yaoshan; Deng, Xianwen; Xie, Zhiqin; Peng, Yi; Wang, Xiuqing

    2012-12-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimized to detect bovine viral diarrhea viral (BVDV) RNA. The RT-LAMP assay is highly sensitive and able to detect 4.67×10(0)copies of BVDV RNA. Additionally, the RT-LAMP method is capable of detecting both genotypes of BVDV. No cross-reaction with other bovine viruses was observed. The ability of RT-LAMP to detect BVDV RNA from bovine fecal swabs was also evaluated. Of the 88 fecal swabs, 38 were found to be positive by RT-LAMP assay, whereas 39 were positive by real-time RT-PCR. Taken together, the BVDV specific RT-LAMP method is highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples.

  10. A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi.

    PubMed

    Britton, Sumudu; Cheng, Qin; Grigg, Matthew J; William, Timothy; Anstey, Nicholas M; McCarthy, James S

    2016-07-01

    The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool.

  11. An integrated microfluidic loop-mediated-isothermal-amplification system for rapid sample pre-treatment and detection of viruses.

    PubMed

    Wang, Chih-Hung; Lien, Kang-Yi; Wang, Ting-Yu; Chen, Tzong-Yueh; Lee, Gwo-Bin

    2011-01-15

    This study presents a novel automatic assay for targeted ribonucleic acid (RNA) extraction and a one-step reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) process for the rapid detection of viruses from tissue samples by utilizing an integrated microfluidic system. By utilizing specific probe-conjugated magnetic beads, target RNA samples can be specifically recognized and hybridized onto the surface of the magnetic beads which are mixed with whole tissue lysates, followed by the synthesis of complementary deoxyribonucleic acid (cDNA) and isothermal amplification of target genes simultaneously with the incorporation of two specific primer sets. The nervous necrosis virus (NNV), the most common aquaculture pathogen, with a mortality rate in infected fish ranging from 80% to 100%, has been selected to verify the performance of the developed miniature system. Experimental results showed that the sensitivity of the integrated microfluidic LAMP system is about 100-fold higher when compared to a conventional one-step reverse-transcript polymerase chain reaction (RT-PCR) process. Significantly, the entire protocol from sample pre-treatment to target gene amplification can be completed within 60 min in an automatic manner without cross-reactions with other tested virus, bacteria and eukaryotic cells. Consequently, this integrated microfluidic LAMP system may provide a powerful platform for rapid purification and detection of virus samples.

  12. Development of a loop-mediated isothermal amplification assay for rapid detection of iridovirus in the Chinese giant salamander.

    PubMed

    Meng, Yan; Zhang, Hui; Liang, Hongwei; Zeng, Lingbing; Xiao, Hanbing; Xie, Congxin

    2013-12-01

    The Chinese giant salamander (Andrias davidianus) iridovirus (GSIV) is an emerging infectious pathogen responsible for severe hemorrhagic disease and high mortality in cultured Chinese giant salamanders. A loop-mediated isothermal amplification (LAMP) assay based on the major caspid protein (MCP) gene has been developed to detect this virus. Primer pairs for the LAMP assay were designed based on the GSIV MCP gene sequence. Amplification results indicate that under optimized conditions the LAMP assay has the ability to specifically detect the virus in both diseased animals and infected epithelioma papilloma cyprinid (EPC) cells. The assay was shown to be 10-fold more sensitive than nested PCR and was able to detect concentrations of 10(-9) (approximately 0.01 pg/μL). The LAMP assay is relatively easy to perform in situ and the amplification products can be observed directly under UV light or via staining with SYBR Green I. The LAMP assay is also rapid and cost-effective. This study establishes the use of a LAMP assay for rapid detection of GSIV, which is a novel and important tool for the diagnosis of GSIV infection in laboratory or farmed Chinese giant salamanders. PMID:24025343

  13. Development of a loop-mediated isothermal amplification assay for rapid detection of iridovirus in the Chinese giant salamander.

    PubMed

    Meng, Yan; Zhang, Hui; Liang, Hongwei; Zeng, Lingbing; Xiao, Hanbing; Xie, Congxin

    2013-12-01

    The Chinese giant salamander (Andrias davidianus) iridovirus (GSIV) is an emerging infectious pathogen responsible for severe hemorrhagic disease and high mortality in cultured Chinese giant salamanders. A loop-mediated isothermal amplification (LAMP) assay based on the major caspid protein (MCP) gene has been developed to detect this virus. Primer pairs for the LAMP assay were designed based on the GSIV MCP gene sequence. Amplification results indicate that under optimized conditions the LAMP assay has the ability to specifically detect the virus in both diseased animals and infected epithelioma papilloma cyprinid (EPC) cells. The assay was shown to be 10-fold more sensitive than nested PCR and was able to detect concentrations of 10(-9) (approximately 0.01 pg/μL). The LAMP assay is relatively easy to perform in situ and the amplification products can be observed directly under UV light or via staining with SYBR Green I. The LAMP assay is also rapid and cost-effective. This study establishes the use of a LAMP assay for rapid detection of GSIV, which is a novel and important tool for the diagnosis of GSIV infection in laboratory or farmed Chinese giant salamanders.

  14. A loop-mediated isothermal amplification (LAMP) method for the identification of species within the Echinococcus granulosus complex.

    PubMed

    Wassermann, Marion; Mackenstedt, Ute; Romig, Thomas

    2014-02-24

    To facilitate the specific identification of Echinococcus spp. isolates in endemic countries, a LAMP (loop-mediated isothermal amplification) assay was developed to detect the various agents known to cause cystic echinococcosis (E. granulosus s.s., E. equinus, E. ortleppi, E. canadensis and E. felidis). The infectivity of the different species and the severity of the disease in humans and livestock vary significantly among those species, and correct molecular identification of large numbers of field isolates is crucial to understand their epidemiology. However, funding constraints in many CE endemic countries often prevent PCR-based screening of field isolates. The LAMP method allows the amplification of DNA fragments under isothermal conditions which can be achieved using an ordinary waterbath, and the detection of amplification products only requires a UV light source. In the present study a LAMP assay was developed which allows the detection and differentiation of the 5 CE causing Echinococcus species. The diagnostic power was adjusted to species level, i.e. intraspecific strains (G1-3 within E. granulosus s.s., G6-10 within E. canadensis) are not discriminated. Wherever this would be necessary for epidemiological purposes, the method can be adjusted according to local requirements. The sensitivity of the assay was tested down to one fiftieth of a single protoscolex or egg, respectively. The present study describes a fast and simple method for the differentiation of CE causing Echinococcus species which can facilitate epidemiological studies in endemic countries.

  15. A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification.

    PubMed

    Chuang, Tsung-Liang; Wei, Shih-Chung; Lee, Szu-Yuan; Lin, Chii-Wann

    2012-02-15

    In this study, we report a simple, low-cost surface plasmon resonance (SPR)-sensing cartridge based on a loop-mediated isothermal amplification (LAMP) method for the on-site detection of the hepatitis B virus (HBV). For LAMP detection, a SPR based LAMP sensing system (SPRLAMP) was constructed, including a novel SPRLAMP sensing cartridge integrating a polymethyl methacrylate (PMMA) micro-reactor with a polycarbonate (PC)-based prism coated with a 50 nm Au film. First, we found that the change of refractive index of the bulk solution was approximately 0.0011 refractive index (RI) units after LAMP reaction. The PC-based prism's linearity and thermal responses were compared to those of a traditional glass prism to show that a PC-based prism can be used for SPR measurement. Finally, the HBV template mixed in the 10 μl LAMP solution could be detected by SPRLAMP system in 17 min even at the detection-limited concentration of 2 fg/ml. We also analyzed the correlation coefficients between the initial concentrations of HBV DNA templates and the system response (ΔRU) at varying amplification times to establish an optimal amplification time endpoint of 25 min (R(2)=0.98). In conclusion, the LAMP reaction could be detected with the SPRLAMP sensing cartridge based on direct sensing of the bulk refractive index.

  16. Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes.

    PubMed

    Hirayama, Hiroki; Kageyama, Soichi; Moriyasu, Satoru; Sawai, Ken; Minamihashi, Akira

    2013-01-01

    In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes.

  17. Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

    PubMed Central

    2010-01-01

    Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic

  18. Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to the infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for a sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to th...

  19. Development of a quantitative loop-mediated isothermal amplification (qLAMP) assay for the detection of Magnaporthe oryzae airborne inoculum in turf ecosystems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grey Leaf Spot (GLS) is a detrimental disease of perennial ryegrass caused by a host-specialized form of Magnaporthe oryzae (Mot). In order to improve turf management, a quantitative loop-mediated isothermal amplification (LAMP) assay coupled with a simple spore trap is being developed to monitor GL...

  20. [Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification].

    PubMed

    Nie, Kai; Wang, Da-Yan; Qin, Meng; Gao, Rong-Bao; Wang, Miao; Zou, Shu-Mei; Han, Feng; Zhao, Xiang; Li, Xi-Yan; Shu, Yue-Long; Ma, Xue-Jun

    2010-03-01

    A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

  1. Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method.

    PubMed

    Huang, Xin; Chen, Lili; Xu, Jiangmin; Ji, Hai-Feng; Zhu, Shuifang; Chen, Hongjun

    2014-08-01

    Transgenic maize plant expressing high phytase activity has been reported and approved by Chinese government in 2009. Here, we report a highly specific loop-mediated isothermal amplification (LAMP) method to detect the phytase gene in the GMO maize. The LAMP reaction takes less than 20min and the amplification is visible without gel electrophoresis. The detection sensitivity of the LAMP method is about 30 copies of phytase genomic DNA, which is 33.3 times greater than the conventional PCR method with gel electrophoresis. The quantitative detection results showed that the LAMP method has a good linear correlation between the DNA copy number and the associated Tt values over a large dynamic range of template concentration from 6×10(1) to 6×10(7) copies, with a quantification limit of 60 copies. Therefore, the LAMP method is visual, faster, and more sensitive, and does not need special equipment compared to traditional PCR technique, which is very useful for field tests and fast screening of GMO feeds. PMID:24629956

  2. Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay.

    PubMed

    Chandra, Amaresh; Keizerweerd, Amber T; Grisham, Michael P

    2016-03-01

    Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection.

  3. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection.

    PubMed

    Nzelu, Chukwunonso O; Gomez, Eduardo A; Cáceres, Abraham G; Sakurai, Tatsuya; Martini-Robles, Luiggi; Uezato, Hiroshi; Mimori, Tatsuyuki; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2014-04-01

    Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)--mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries. PMID:24388795

  4. Development and Evaluation of a Novel and Rapid Detection Assay for Botrytis cinerea Based on Loop-Mediated Isothermal Amplification

    PubMed Central

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63°C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10−3 ng µL−1 of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10−2 ng µL−1). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables. PMID:25329402

  5. Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay.

    PubMed

    Chandra, Amaresh; Keizerweerd, Amber T; Grisham, Michael P

    2016-03-01

    Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection. PMID:26837389

  6. Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk.

    PubMed

    Bosward, Katrina L; House, John K; Deveridge, Amber; Mathews, Karen; Sheehy, Paul A

    2016-03-01

    Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen. PMID:26778303

  7. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection.

    PubMed

    Nzelu, Chukwunonso O; Gomez, Eduardo A; Cáceres, Abraham G; Sakurai, Tatsuya; Martini-Robles, Luiggi; Uezato, Hiroshi; Mimori, Tatsuyuki; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2014-04-01

    Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)--mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.

  8. A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

    PubMed

    Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2016-12-15

    Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC). PMID:27393827

  9. Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay.

    PubMed

    Liu, Aihong; Guan, Guiquan; Du, Pengfei; Gou, Huitian; Zhang, Jun; Liu, Zhijie; Ma, Milin; Ren, Qiaoyun; Liu, Junlong; Yang, Jifei; Li, Youquan; Niu, Qinli; Bai, Qi; Yin, Hong; Luo, Jianxun

    2013-01-16

    The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species -T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55 min or 50 min under isothermal conditions of 61°C or 63°C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species -T. sergenti and T. sinensis, especially in endemic countries.

  10. Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene

    PubMed Central

    Wang, Yi; Wang, Yan; Xu, Huaqing; Dai, Hang; Meng, Shuang; Ye, Changyun

    2014-01-01

    A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories. PMID:25549337

  11. A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry

    PubMed Central

    Wang, Hehe; Turechek, William W.

    2016-01-01

    Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×103 CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2–3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management. PMID:26766068

  12. Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method.

    PubMed

    Huang, Xin; Chen, Lili; Xu, Jiangmin; Ji, Hai-Feng; Zhu, Shuifang; Chen, Hongjun

    2014-08-01

    Transgenic maize plant expressing high phytase activity has been reported and approved by Chinese government in 2009. Here, we report a highly specific loop-mediated isothermal amplification (LAMP) method to detect the phytase gene in the GMO maize. The LAMP reaction takes less than 20min and the amplification is visible without gel electrophoresis. The detection sensitivity of the LAMP method is about 30 copies of phytase genomic DNA, which is 33.3 times greater than the conventional PCR method with gel electrophoresis. The quantitative detection results showed that the LAMP method has a good linear correlation between the DNA copy number and the associated Tt values over a large dynamic range of template concentration from 6×10(1) to 6×10(7) copies, with a quantification limit of 60 copies. Therefore, the LAMP method is visual, faster, and more sensitive, and does not need special equipment compared to traditional PCR technique, which is very useful for field tests and fast screening of GMO feeds.

  13. Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene.

    PubMed

    Wei, Xiuying; Shi, Xingming; Zhao, Yan; Zhang, Jing; Wang, Mei; Liu, Changjun; Cui, Hongyu; Hu, Shunlei; Quan, Yanming; Chen, Hongyan; Wang, Yunfeng

    2012-08-01

    A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

  14. Rapid detection of human rotavirus using NSP4 gene specific reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Malik, Yashpal Singh; Sharma, Kuldeep; Kumar, Naveen; Shivachandra, Sathish B; Rawat, Vinita; Rakholia, Ritu; Ranjan, Rajeev; Ganesh, Balasubramanian; Parida, Manmohan

    2013-09-01

    The seasonal outbreaks of human rotavirus (RV) infection occur every winter. Most patients are diagnosed clinically by a rapid latex agglutination detection kit or polymerase chain reaction assays for RV from stool samples, but some problems have been reported on the specificity and sensitivity of such rapid detection assays. To ratify these issues, a sensitive, specific, simple, and rapid nucleic acid based diagnostic method is expected to be introduced and the reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the RV in human stool samples by incubation at 60 °C for 1 h and amplification was confirmed by electrophoretic laddering, restriction enzyme digestion, and hydroxynapthol blue discoloration. The assay established in this study was found to detect only the RVs and no cross-reaction with other viruses, demonstrating its high specificity. By using serial samples dilution as template, the detection limit of LAMP was 10 times more than that of PCR. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RV with high sensitivity in comparison to conventional RT-PCR.

  15. Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method.

    PubMed

    Liu, Zhongyuan; Dong, Zhijun; Liu, Dongyan

    2016-07-01

    Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle.

  16. Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

    PubMed

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Pradeep, Padmaja Jayaprasad; Jitrakorn, Sarocha; Arunrut, Narong; Sangsuriya, Pakkakul; Saksmerprome, Vanvimon; Senapin, Saengchan; Withyachumnarnkul, Boonsirm; Kiatpathomchai, Wansika

    2015-12-01

    Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria. PMID:26648105

  17. Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients.

    PubMed

    Singh, Preeti; Singh, Sundeep; Mirdha, Bijay Ranjan; Guleria, Randeep; Agarwal, Sanjay Kumar; Mohan, Anant

    2015-01-01

    Pneumocystis pneumonia (PCP) is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%), 41/185 (22.2%), and 49/185 (26.5%) samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p < 0.05) with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection. PMID:26664746

  18. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    PubMed

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables. PMID:25329402

  19. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    PubMed

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV. PMID:23933076

  20. Loop-mediated isothermal amplification (LAMP) assay for detection of Theileria sergenti infection targeting the p33 gene.

    PubMed

    Wang, L X; He, L; Fang, R; Song, Q Q; Tu, P; Jenkins, A; Zhou, Y Q; Zhao, J L

    2010-07-15

    Theileria is a widespread, intraerythrocytic tick-borne protozoan parasite. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method offering rapid, accurate and cost-effective diagnosis of infectious diseases. In this study, the LAMP method was developed for detecting Theileria sergenti. Four primers were designed from six distinct regions of the target gene, a 33-kDa major piroplasm surface protein (p33) gene in T. sergenti. The specificity assay showed it was specific for T. sergenti whilst the LAMP was able to detect a parasitemia level of 0.000002% which was more sensitive than conventional PCR. 154 field samples from water buffalo and 159 field samples from cattle were analyzed using the LAMP method. About 60.4% (96/159) of cattle samples were positive by LAMP, compared to 30.0% (46/154) of water buffalo samples that were positive. Compared with conventional PCR, the LAMP method exhibited higher detection abilities than conventional PCR. All the results indicated that the LAMP assay is a simple and convenient diagnostic tool for theileriosis.

  1. Detection of shrimp Taura syndrome virus by loop-mediated isothermal amplification using a designed portable multi-channel turbidimeter.

    PubMed

    Sappat, Assawapong; Jaroenram, Wansadaj; Puthawibool, Teeranart; Lomas, Tanom; Tuantranont, Adisorn; Kiatpathomchai, Wansika

    2011-08-01

    In this study, a portable turbidimetric end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) using spectroscopic measurement of a loop-mediated isothermal amplification (LAMP) by-product: magnesium pyrophosphate (Mg(2)P(2)O(7)). The device incorporated a heating block that maintained an optimal temperature of 63°C for the duration of the RT-LAMP reaction. Turbidity of the RT-LAMP by-product was measured when light from a light-emitting diode (LED) passed through the tube to reach a light dependent resistance (LDR) detector. Results revealed that turbidity measurement of the RT-LAMP reactions using this device provided the same detection sensitivity as the agarose gel electrophoresis detection of RT-LAMP and nested RT-PCR (IQ2000™) products. Cross reactions with other shrimp viruses were not found, indicating that the RT-LAMP-turbidity measurement was highly specific to TSV. The combination of 10 min for rapid RNA preparation with 30 min for RT-LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1h compared to 4-8h for the nested RT-PCR method. RT-LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of TSV in the field.

  2. Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus.

    PubMed

    Dauner, Allison L; Mitra, Indrani; Gilliland, Theron; Seales, Sajeewane; Pal, Subhamoy; Yang, Shih-Chun; Guevara, Carolina; Chen, Jiann-Hwa; Liu, Yung-Chuan; Kochel, Tadeusz J; Wu, Shuenn-Jue L

    2015-09-01

    During dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. To facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that detects all 4 serotypes of dengue virus (DENV). We used a quencher to reduce nonspecific amplification. The assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at 63 °C. Results can be visualized using UV fluorescence, handheld readers, or lateral flow immunochromatographic tests. We report a sensitivity of 86.3% (95% confidence interval [CI], 72.7-94.8%) and specificity of 93.0% (95% CI, 83.0-98.1%) using a panel of clinical specimens characterized by DENV quantitative reverse transcription-polymerase chain reaction. This pan-serotype DENV RT-LAMP can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need.

  3. Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification.

    PubMed

    Sun, Zhao-Feng; Hu, Chao-Qun; Ren, Chun-Hua; Shen, Qi

    2006-01-01

    A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome DNA. By the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by the white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. The assay had a detection limit of 5-500 copies of DNA template with gel electrophoresis, SYBR Green I and white turbidity with naked-eye inspection. The detection sensitivity of LAMP was 100-fold higher than the PCR. A diagnostic procedure which is rapid and highly sensitive was developed for IHHNV detection.

  4. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    PubMed

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

  5. Rapid and Real-Time Detection of Chikungunya Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Assay▿

    PubMed Central

    Parida, M. M.; Santhosh, S. R.; Dash, P. K.; Tripathi, N. K.; Lakshmi, V.; Mamidi, N.; Shrivastva, A.; Gupta, N.; Saxena, P.; Babu, J. Pradeep; Rao, P. V. Lakshmana; Morita, Kouichi

    2007-01-01

    The standardization and validation of a one-step, single-tube, accelerated, quantitative reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene for the rapid and real-time detection of Chikungunya virus (CHIKV) are reported. A linear relationship between the amount of template and time of positivity value over a range of 2 × 108 to 2 × 102 copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The comparative evaluation of the RT-LAMP assay with acute-phase patient serum samples demonstrated exceptionally higher sensitivity by correctly identifying 21 additional positive borderline cases that were missed by conventional RT-PCR (P < 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient serum samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2 × 108 to 2 × 101 copies. In addition, the field applicability of the RT-LAMP assay was also demonstrated by standardizing SYBR Green I-based RT-LAMP wherein the amplification was carried out in a water bath at 63°C for 60 min, which was followed by monitoring gene amplification with the naked eye through color changes. These findings demonstrated that the RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute-phase serum samples without requiring any sophisticated equipment and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries. PMID:17135444

  6. Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection

    PubMed Central

    2011-01-01

    Background Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. Methods A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. Results The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method. Conclusions This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings. PMID:21693031

  7. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    PubMed

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  8. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use

    PubMed Central

    2012-01-01

    Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must

  9. Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

    NASA Astrophysics Data System (ADS)

    Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

    2015-02-01

    A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

  10. Rapid detection of nusG and fadA in Fusobacterium nucleatum by loop-mediated isothermal amplification.

    PubMed

    Huang, Simo; Yang, Zhan; Zou, Dayang; Dong, Derong; Liu, Anheng; Liu, Wei; Huang, Liuyu

    2016-08-01

    Fusobacterium nucleatum is associated with various human diseases such as periodontal disease and colorectal cancer (CRC); thus, F. nucleatum detection might serve as a novel diagnostic tool. Here, we describe the development of a sensitive and rapid molecular method for detecting two F. nucleatum genes: the highly conserved nusG and fadA, which encode a critical host colonization factor. Loop-mediated isothermal amplification (LAMP) primer sets for the rapid detection of nusG and fadA were designed and optimized. The nusG primers yielded consistent negative results for 20 non-F. nucleatum bacterial strains, confirming the high specificity of the primers. LAMP reaction primer sensitivity was determined, and its detection rate in comparison to conventional PCR was assessed using 57 clinical stool samples. The LAMP detection limit for nusG and fadA was 22.5 and 0.225 pg µl-1, respectively, indicating that the sensitivity of this method was 10-fold higher than that of conventional PCR. These results suggest that the LAMP technique is able to effectively identify F. nucleatum via nusG as well as detect its virulence factor. To the best of our knowledge, this study is the first to report the application of LAMP for the detection of nusG and fadA in F. nucleatum. The LAMP method constitutes a sensitive and specific visual assay for the rapid detection of the pathogen F. nucleatum.

  11. Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

    PubMed Central

    Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  12. Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the sexually-transmitted parasite, Trichomonas vaginalis.

    PubMed

    Adao, Davin Edric V; Rivera, Windell L

    2016-01-01

    A loop-mediated isothermal amplification (LAMP) assay was developed to detect the sexually-transmitted parasite, Trichomonas vaginalis in vaginal swabs. The presence of T. vaginalis was detected from 121 female sex workers attending a social hygiene clinic in Balibago, Angeles City, Pampanga, Philippines using culture, polymerase chain reaction (PCR), and the developed LAMP assay. The high analytical sensitivity of LAMP detected a higher prevalence of T. vaginalis (42.06%) compared to culture (8.26%) and PCR (7.44%). Additionally, this assay did not cross-react with DNAs of other trichomonads that can infect humans such as Trichomonas tenax and Pentatrichomonas hominis as well as the pathogens, Candida albicans and Staphylococcus aureus. The LAMP assay developed had a limit of detection (0.036 ng/μl) lower than that of PCR using the primers TvK3 and TvK7 (0.36 ng/μl). Prevalence of T. vaginalis in female sex workers in this area of the Philippines may be higher than previously estimated. Discordant results of PCR and LAMP may be due to different reactions to different kinds of inhibitors in the vaginal swabs. PMID:27262954

  13. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    PubMed

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.

  14. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening. PMID:24188249

  15. Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes.

    PubMed

    Thekisoe, Oriel M M; Rambritch, Natasha E; Nakao, Ryo; Bazie, Raoul S; Mbati, Peter; Namangala, Boniface; Malele, Imna; Skilton, Robert A; Jongejan, Frans; Sugimoto, Chihiro; Kawazu, Shin-Ichiro; Inoue, Noboru

    2010-01-01

    We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.

  16. Rapid sex identification of papaya (Carica papaya) using multiplex loop-mediated isothermal amplification (mLAMP).

    PubMed

    Hsu, Te-Hua; Gwo, Jin-Chywan; Lin, Kuan-Hung

    2012-10-01

    Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (<1 h), isothermal conditions (less equipment required), a high efficiency (0.5 ng of DNA required in the reaction mixture), and an economical reaction system (5 μl in volume). The established method can be easily performed in the field by visual inspection and facilitates the selection of all hermaphroditic individuals in papaya production.

  17. Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas

    PubMed Central

    Chen, Xiaolu; Ma, Lijuan; Qiang, Song; Ma, Deying

    2016-01-01

    Ascochyta blight (AB) is a devastating fungal disease of chickpeas that has spread to nearly all of the chickpea cultivating regions of the world. The rapid diagnosis of Ascochyta rabiei L. (A. rabiei), the pathogen that causes AB, plays an important role in A. rabiei epidemic tracking and AB management. In this study, a group of loop-mediated isothermal amplification (LAMP) primers was designed to detect A. rabiei in chickpea plants and seeds via a LAMP method and a conventional PCR method based on an internal transcribed spacer (ITS) sequence analysis of A. rabiei. Compared with the conventional PCR method, the LAMP method not only exhibited greater sensitivity and specificity in the detection of A. rabiei but also used simpler equipment and required less operational time. The minimum detectable concentration of the A. rabiei genomic DNA solution with the LAMP method was 6.01 × 10−6 ng/μl, which was 100 times lower than that of the conventional PCR method with the same outer primers. The greatest advantage of the LAMP method is that results can be observed via the visualization of color changes in SYBR Green I dye with the naked eye, and it does not require expensive instruments, also with less time consumption. PMID:27161564

  18. Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes.

    PubMed

    Thekisoe, Oriel M M; Rambritch, Natasha E; Nakao, Ryo; Bazie, Raoul S; Mbati, Peter; Namangala, Boniface; Malele, Imna; Skilton, Robert A; Jongejan, Frans; Sugimoto, Chihiro; Kawazu, Shin-Ichiro; Inoue, Noboru

    2010-01-01

    We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries. PMID:19654009

  19. Development of reverse transcription loop-mediated isothermal amplification assays to detect Hantaan virus and Seoul virus.

    PubMed

    Hu, Dan; Hao, Lina; Zhang, Jinhai; Yao, Pingping; Zhang, Qi; Lv, Heng; Gong, Xiufang; Pan, Xiuzhen; Cao, Min; Zhu, Jin; Zhang, Yun; Feng, Youjun; Wang, Changjun

    2015-09-01

    We developed two assays based on one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) to identify Hantaan virus (HTNV) and Seoul virus (SEOV), members of the Hantavirus genus that cause hemorrhagic fever with renal syndrome (HFRS). Our results showed that these assays can be conducted within 30min under isothermal conditions. The detection limit for HTNV was around 10 copies per reaction, similar to detection levels for quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. The detection limit for SEOV was 100 copies per reaction, a sensitivity that was 10-fold lower than that for qRT-PCR assays but 10-fold higher than that for RT-PCR assays. The method we developed was specific for both HTNV and SEOV without any cross-reaction with other pathogens. We conclude that RT-LAMP assays could be useful for the rapid and direct detection of HTNV and SEOV clinically, and for the epidemiological investigation of HFRS.

  20. Loop mediated isothermal amplification combined with nucleic acid lateral flow strip for diagnosis of cyprinid herpes virus-3.

    PubMed

    Soliman, Hatem; El-Matbouli, Mansour

    2010-02-01

    An improved loop mediated isothermal amplification (LAMP) assay for rapid, sensitive and specific detection of cyprinid herpes virus-3 (CyHV-3), also known as koi herpes virus (KHV), was developed. The lower detection limit of the CyHV-3-LAMP assay is 10 fg DNA which equivalent to 30 copies of CyHV-3 genome. Nucleic acid lateral flow assay was used for visual detection of the LAMP products. The LAMP- nucleic acid lateral flow assay relies on DNA hybridization technology and antigen-antibody reactions in combination with LAMP. For application of this assay, the biotinylated LAMP product was hybridized with a FITC-labelled specific probe for 5 min. The resulting DNA complex could be visualised as purple band at the strip test line within 5 min of sample exposure. The nucleic acid lateral flow analysis of the LAMP product was equivalent in sensitivity but more rapid than the conventional agarose gel electrophoresis. The combination of LAMP assay with the nucleic acid lateral flow analysis can simplify the diagnosis and screening of CyHV-3 as it is simple, requires very little training, does not require specialized equipment such as a thermal cycler, the results are read visually with no need to run a gel and has a high sensitivity and specificity.

  1. Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

    PubMed Central

    Tang, Qing; Tian, Shuguang; Yu, Nong; Zhang, Xi; Jia, Xiaodong; Zhai, Hongyan

    2016-01-01

    Aspergillus fumigatus is a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection of A. fumigatus infection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection of A. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatus strains, including 5 species of the Aspergillus genus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 102 copies for the same target. Clinical samples from a total of 69 patients with probable IA (n = 14) and possible IA (n = 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection of A. fumigatus in clinical testing has been developed. PMID:26791368

  2. Real-time fluorescence Loop-Mediated Isothermal Amplification (LAMP) for rapid and reliable diagnosis of pulmonary tuberculosis.

    PubMed

    Cao, Donglin; Hu, Liangshan; Lin, Maorui; Li, Mingyou; Ye, Zebing; Sun, Hongtao; Huang, Jiwei; Yang, Huawen; Tian, Junzhang

    2015-02-01

    A reliable, simple and rapid diagnostic method that can be helpful in pulmonary tuberculosis diagnosis is urgently needed. Loop-mediated Isothermal Amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. In this study, real-time fluorescence LAMP was evaluated to rapidly detect Mycobacterium tuberculosis in sputum and was compared to the performance of real-time fluorescence quantitative PCR (Q-PCR). All the standard MTB strains were successfully detected and limit of detection (LOD) was 10(2)CFU/mL by real-time fluorescence LAMP within 20min. In light of MTB in sputum, the real-time fluorescence LAMP method yielded a sensitivity of 98.0% and a specificity of 78.3%, compared to Q-PCR assay, which yielded a sensitivity of 96.0% and a specificity of 82.6% for PTB diagnosis. There was an excellent overall agreement between LAMP and Q-PCR for PTB (κ=0.315) and non-PTB (κ=0.862). Therefore, the real-time fluorescence LAMP assay is a rapid, sensitive, and specific method to detect pulmonary tuberculosis.

  3. Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas.

    PubMed

    Chen, Xiaolu; Ma, Lijuan; Qiang, Song; Ma, Deying

    2016-01-01

    Ascochyta blight (AB) is a devastating fungal disease of chickpeas that has spread to nearly all of the chickpea cultivating regions of the world. The rapid diagnosis of Ascochyta rabiei L. (A. rabiei), the pathogen that causes AB, plays an important role in A. rabiei epidemic tracking and AB management. In this study, a group of loop-mediated isothermal amplification (LAMP) primers was designed to detect A. rabiei in chickpea plants and seeds via a LAMP method and a conventional PCR method based on an internal transcribed spacer (ITS) sequence analysis of A. rabiei. Compared with the conventional PCR method, the LAMP method not only exhibited greater sensitivity and specificity in the detection of A. rabiei but also used simpler equipment and required less operational time. The minimum detectable concentration of the A. rabiei genomic DNA solution with the LAMP method was 6.01 × 10(-6 )ng/μl, which was 100 times lower than that of the conventional PCR method with the same outer primers. The greatest advantage of the LAMP method is that results can be observed via the visualization of color changes in SYBR Green I dye with the naked eye, and it does not require expensive instruments, also with less time consumption. PMID:27161564

  4. Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees.

    PubMed

    Ptaszyńska, Aneta A; Borsuk, Grzegorz; Woźniakowski, Grzegorz; Gnat, Sebastian; Małek, Wanda

    2014-08-01

    Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae.

  5. Evaluation of Loop-Mediated Isothermal Amplification Suitable for Molecular Monitoring of Schistosome-Infected Snails in Field Laboratories

    PubMed Central

    Hamburger, Joseph; Abbasi, Ibrahim; Kariuki, Curtis; Wanjala, Atsabina; Mzungu, Elton; Mungai, Peter; Muchiri, Eric; King, Charles H.

    2013-01-01

    We previously described loop-mediated isothermal amplification (LAMP) for detection of Schistosoma haematobium and S. mansoni DNA in infected snails. In the present study, we adapted the LAMP assay for application in field laboratories in schistosomiasis-endemic areas. Isolation of DNA was simplified by blotting snail tissue (extracted in NaOH/sodium dodecyl sulfate) onto treated membranes, which enabled preservation at ambient temperatures. A ready-mix of LAMP reagents, suitable for shipment at ambient temperature and storage in minimal refrigeration, was used. Local survey teams without experience in molecular biology acquired operational expertise with this test within a few hours. Fifty-four field-caught snails were tested locally by LAMP and 59 were tested at similar conditions in Jerusalem. The LAMP results were consistent with those of a polymerase chain reaction; only four samples showed false-negative results. Results indicate that LAMP assays are suitable for detection of S. haematobium and S. mansoni in low-technology parasitology laboratories in which schistosomiasis elimination activities are undertaken. PMID:23208875

  6. Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

    PubMed Central

    Zhou, Dinggang; Guo, Jinlong; Xu, Liping; Gao, Shiwu; Lin, Qingliang; Wu, Qibin; Wu, Luguang; Que, Youxiong

    2014-01-01

    To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg2+, 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn2+ (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane. PMID:24810230

  7. Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus.

    PubMed

    Tang, Qing; Tian, Shuguang; Yu, Nong; Zhang, Xi; Jia, Xiaodong; Zhai, Hongyan; Sun, Qun; Han, Li

    2016-04-01

    Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10(2)copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed.

  8. Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis.

    PubMed

    Kitano, Taiichi; Mikami, Yoshikazu; Iwase, Takashi; Asano, Masatake; Komiyama, Kazuo

    2016-01-01

    Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P. gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P. gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P. gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P. gingivalis was localized in a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P. gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P. gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P. gingivalis in the progression of periapical periodontitis. (J Oral Sci 58, 163-169, 2016).

  9. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    PubMed

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection. PMID:26892741

  10. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Lenarčič, Rok; Morisset, Dany; Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  11. A Novel Loop-Mediated Isothermal Amplification Assay for Serogroup Identification of Neisseria meningitidis in Cerebrospinal Fluid

    PubMed Central

    Lee, DoKyung; Kim, Eun Jin; Kilgore, Paul E.; Takahashi, Hideyuki; Ohnishi, Makoto; Tomono, Jun; Miyamoto, Shigehiko; Omagari, Daisuke; Kim, Dong Wook; Seki, Mitsuko

    2016-01-01

    We have developed a novel Neisseria meningitidis serogroup-specific loop-mediated isothermal amplification (LAMP) assay for six of the most common meningococcal serogroups (A, B, C, W, X, and Y). The assay was evaluated using a set of 31 meningococcal LAMP assay positive cerebrospinal fluid (CSF) specimens from 1574 children with suspected meningitis identified in prospective surveillance between 1998 and 2002 in Vietnam, China, and Korea. Primer specificity was validated using 15 N. meningitidis strains (including serogroups A, B, C, E, W, X, Y, and Z) and 19 non-N. meningitidis species. The N. meningitidis serogroup LAMP detected down to ten copies and 100 colony-forming units per reaction. Twenty-nine CSF had N. meningitidis serogroup identified by LAMP compared with two CSF in which N. meningitidis serogroup was identified by culture and multi-locus sequence typing. This is the first report of a serogroup-specific identification assay for N. meningitidis using the LAMP method. Our results suggest that this assay will be a rapid, sensitive, and uniquely serogroup-specific assay with potential for application in clinical laboratories and public health surveillance systems. PMID:26793181

  12. Meningococcal carriage rates in healthy individuals in Japan determined using Loop-Mediated Isothermal Amplification and oral throat wash specimens.

    PubMed

    Takahashi, Hideyuki; Haga, Masae; Sunagawa, Tomimasa; Saitoh, Takehito; Kitahara, Takeru; Matsumoto, Sohkichi; Ohnishi, Makoto

    2016-07-01

    The detailed epidemiology of meningococcal diseases in Japan has yet to be determined and, moreover, the healthy carriage rate is also unknown. In this study, to obtain insight into the carriage rate of Neisseria meningitidis in healthy individuals in Japan, we developed a new method to detect the N. meningitidis-specific ctrB gene, one of the genes encoding enzymes for capsule synthesis, by Loop-Mediated Isothermal Amplification (LAMP) and examined the meningococcal carriage rate by using self-collected oral throat wash specimens from 836 students at a university. Examination by LAMP showed that 7 out of 836 samples were positive for N. meningitidis DNA, and the results were also verified by the nested PCR method for the meningococcus specific ggt gene. The N. meningitidis carriage rate in healthy individuals was estimated to be 0.84%. Moreover, we further confirmed by the nested-PCR-based serogroup typing method that 5 of the positive samples belonged to serogroup Y, 1 belonged to group B and 1 was unidentifiable. Considering the epidemiology for meningococcal diseases in Japan, the carriage rate and the serogroup profile seem to be consistent with low incidence of meningococcal diseases and serogroup distribution of clinical meningococcal isolates in Japan, respectively. PMID:26895673

  13. Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence.

    PubMed

    Wang, Jiuhai; Lu, Peng; Yan, Jieni; Zhang, Yufan; Huang, Lanye; Ali, Zeeshan; Li, Zhiyang; He, Nongyue

    2016-04-01

    RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus. PMID:27301197

  14. Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat.

    PubMed

    Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

    2014-01-01

    Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

  15. Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method

    PubMed Central

    Srisawat, Mevaree; Panbangred, Watanalai

    2015-01-01

    The Salmonella enterotoxin (stn) gene exhibits high homology among S. enterica serovars and S. bongori. A set of 6 specific primers targeting the stn gene were designed for detection of Salmonella spp. using the loop-mediated isothermal amplification (LAMP) method. The primers amplified target sequences in all 102 strains of 87 serovars of Salmonella tested and no products were detected in 57 non-Salmonella strains. The detection limit in pure cultures was 5 fg DNA/reaction when amplified at 65°C for 25 min. The LAMP assay could detect Salmonella in artificially contaminated food samples as low as 220 cells/g of food without a preenrichment step. However, the sensitivity was increased 100-fold (~2 cells/g) following 5 hr preenrichment at 35°C. The LAMP technique, with a preenrichment step for 5 and 16 hr, was shown to give 100% specificity with food samples compared to the reference culture method in which 67 out of 90 food samples gave positive results. Different food matrixes did not interfere with LAMP detection which employed a simple boiling method for DNA template preparation. The results indicate that the LAMP method, targeting the stn gene, has great potential for detection of Salmonella in food samples with both high specificity and high sensitivity. PMID:26543859

  16. A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex.

    PubMed

    Hong, Ming; Zha, Lei; Fu, Wenliang; Zou, Minji; Li, Wuju; Xu, Donggang

    2012-02-01

    This study was aimed to rapidly identify and differentiate two main pathogens of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium bovis by a modified loop-mediated isothermal amplification (LAMP) assay. The reaction results could be evaluated by naked eye with two optimized closed tube detection methods as follows: adding the modified fluorescence dye in advance into the reaction mix so as to observe the color changes or putting a tinfoil in the tube and adding the SYBR Green I dye on it, then making the dye drop into the bottom of the tube by centrifuge after reaction. The results showed that the two groups of primers used jointly in this assay could successfully identify and differentiate Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium tuberculosis bovis. Sensitivity test displayed that the modified LAMP assay with the closed tube system could determine the minimal template concentration of 1 copy/μl, which was more sensitive than that of routine PCR. The advantages of this LAMP method for detection of the Mycobacterium tuberculosis complex included high specificity, high sensitivity, simplicity, and superiority in avoidance of aerosol contamination. The modified LAMP assay would provide a potential for clinical diagnosis and therapy of tuberculosis in the developing countries and the resource-limited areas. PMID:22806847

  17. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening.

  18. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat

    PubMed Central

    Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

    2014-01-01

    Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

  19. Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient Hedyotis diffusa Willd.

    PubMed

    Li, Ming; Wong, Yuk-Lau; Jiang, Li-Li; Wong, Ka-Lok; Wong, Yuen-Ting; Lau, Clara Bik-San; Shaw, Pang-Chui

    2013-12-01

    Hedyotis diffusa Willd. (Baihuasheshecao) is an ingredient of herbal teas commonly consumed in the Orient and tropical Asia for cancer treatment and health maintenance. In the market, this ingredient is frequently adulterated by the related species Hedyotis corymbosa (L.) Lam. The objective of this study is to develop a novel loop-mediated isothermal amplification (LAMP) technique to differentiate H. diffusa from its adulterant H. corymbosa. A set of four internal control primers (F3, FIP, BIP and B3) were designed based on six loci in the internal transcribed spacer (ITS) for LAMP of both H. diffusa and H. corymbosa. Two specific primers (S_F3 and S_FIP) were designed for specific LAMP detection of H. diffusa only. Our data showed that LAMP was successful for both H. diffusa and H. corymbosa in internal control. In contrast, only H. diffusa was detected in specific LAMP using the specific primers S_F3 and S_FIP. This study showed that LAMP was useful to differentiate H. diffusa from its adulterant H. corymbosa. This study is significant for the verification of the authenticity for better quality control of this common herbal tea ingredient. The strategy of including an internal control assures the quality of the concerned DNA region for LAMP.

  20. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model.

    PubMed

    Ngotho, Maina; Kagira, John Maina; Gachie, Beatrice Muthoni; Karanja, Simon Muturi; Waema, Maxwell Wambua; Maranga, Dawn Nyawira; Maina, Naomi Wangari

    2015-01-01

    Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

  1. A portable automatic endpoint detection system for amplicons of loop mediated isothermal amplification on microfluidic compact disk platform.

    PubMed

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10(-3) ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  2. Detection of Trypanosoma cruzi and T. rangeli Infections from Rhodnius pallescens Bugs by Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Thekisoe, Oriel M. M.; Rodriguez, Carol V.; Rivas, Francisco; Coronel-Servian, Andrea M.; Fukumoto, Shinya; Sugimoto, Chihiro; Kawazu, Shin-Ichiro; Inoue, Noboru

    2010-01-01

    We have developed two loop-mediated isothermal amplification (LAMP) assays for specific detection of Trypanosoma cruzi and Trypanosoma rangeli based on the 18S ribosomal RNA (rRNA) and the small nucleolar RNA (snoRNA) genes, respectively. The detection limit of the assays is 100 fg and 1 pg for T. cruzi and T. rangeli, respectively, with reactions conducted in 60 minutes. The two LAMP assays were used in detection of T. cruzi and T. rangeli infections in comparison with polymerase chain reaction (PCR) for DNA samples extracted from Rhodnius pallescens bugs collected from palm trees in Panama. Out of a total of 52 DNA samples from R. pallescens bugs 17 (33%) and 14 (27%) were T. cruzi-positive by LAMP and PCR, respectively, while, 7 (13%) and 4 (8%) were T. rangeli-positive by LAMP and PCR, respectively. Further evaluation of these LAMP assays is needed, especially with specimens collected from human patients as well as blood kept for transfusion purposes. PMID:20439966

  3. Graphene oxide based fluorescence resonance energy transfer and loop-mediated isothermal amplification for white spot syndrome virus detection.

    PubMed

    Waiwijit, U; Phokaratkul, D; Kampeera, J; Lomas, T; Wisitsoraat, A; Kiatpathomchai, W; Tuantranont, A

    2015-10-20

    Graphene oxide (GO) is attractived for biological or medical applications due to its unique electrical, physical, optical and biological properties. In particular, GO can adsorb DNA via π-π stacking or non-covalent interactions, leading to fluorescence quenching phenomenon applicable for bio-molecular detection. In this work, a new method for white spot syndrome virus (WSSV)-DNA detection is developed based on loop-mediated isothermal amplification (LAMP) combined with fluorescence resonance energy transfer (FRET) between GO and fluorescein isothiocyanate-labeled probe (FITC-probe). The fluorescence quenching efficiency of FITC-probe was found to increase with increasing GO concentration and reached 98.7% at a GO concentration of 50 μg/ml. The fluorescence intensity of FITC-probe was recovered after hybridization with WSSV LAMP product with an optimal hybridization time of 10 min and increased accordingly with increasing amount of LAMP products. The detection limit was estimated to be as low as 10 copies of WSSV plasmid DNA or 0.6 fg of the total DNA extracted from shrimp infected with WSSV. In addition, no cross reaction was observed with other common shrimp viral pathogens. Therefore, the GO-FRET-LAMP technique is promising for fast, sensitive and specific detection of DNAs. PMID:26277651

  4. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

    PubMed Central

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S.; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  5. Development of phage immuno-loop-mediated isothermal amplification assays for organophosphorus pesticides in agro-products.

    PubMed

    Hua, Xiude; Yin, Wei; Shi, Haiyan; Li, Ming; Wang, Yanru; Wang, Hong; Ye, Yonghao; Kim, Hee Joo; Gee, Shirley J; Wang, Minghua; Liu, Fengquan; Hammock, Bruce D

    2014-08-19

    Two immuno-loop-mediated isothermal amplification assays (iLAMP) were developed by using a phage-borne peptide that was isolated from a cyclic eight-peptide phage library. One assay was used to screen eight organophosphorus (OP) pesticides with limits of detection (LOD) between 2 and 128 ng mL(-1). The iLAMP consisted of the competitive immuno-reaction coupled to the LAMP reaction for detection. This method provides positive results in the visual color of violet, while a negative response results in a sky blue color; therefore, the iLAMP allows one to rapidly detect analytes in yes or no fashion. We validated the iLAMP by detecting parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery, and the detection results were consistent with the enzyme-linked immunosorbent assay (ELISA). In conclusion, the iLAMP is a simple, rapid, sensitive, and economical method for detecting OP pesticide residues in agro-products with no instrumental requirement. PMID:25135320

  6. Loop-mediated isothermal amplification (LAMP) detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758) in China.

    PubMed

    He, Lan; Zhou, Yan-Qin; Oosthuizen, Marinda C; Zhao, Jun-Long

    2009-10-28

    Loop-mediated isothermal amplification (LAMP) is a rapid method with high specificity and efficiency under isothermal condition using a set of four specifically designed primers that recognize six distinct sequences on the target gene. In this study, a LAMP method was developed for specific detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758). Four primers were designed from the V4 hypervariable region of the 18S rRNA gene of B. orientalis. Blood samples were collected from B. orientalis experimentally infected water buffalo as well as from 165 water buffalo from eight different regions of the Hubei province, south China. Genomic DNA was extracted, subjected to the LAMP assay and compared with results obtained using a previously described semi-nested PCR. The LAMP assay proofed to be B. orientalis specific and more sensitive than the semi-nested PCR. While previously B. orientalis had not been reported north of the Yangtse River, our results show that B. orientalis has spread to the north of the river. This could pose a serious threat to the water buffalo industry.

  7. Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives.

    PubMed

    Biswas, Gouranga; Sakai, Masahiro

    2014-04-01

    Since its invention in 2000, loop-mediated isothermal amplification (LAMP) assay has been one of the most extensively used molecular diagnostic tools in bio-medical fields due to the rapidity, accuracy, and cost-effectiveness of the technique. This technique has also earned popularity in aquaculture disease diagnosis. Aquaculture, as a result of its rapid intensification and expansion, experiences increased infectious disease occurrences. For maintenance of economic viability, rapid, sensitive and efficient diagnosis of disease causing agents is an important step prior to undertaking effective prevention and control measures in aquaculture. Constraints on time and expertise required for conventional biochemical, serological and polymerase chain reaction (PCR)-based techniques offer avenues in adoption of the LAMP by the aquaculturists at field conditions. This assay has been successfully applied in detection of several bacterial, viral and parasitic pathogens causing serious diseases in aquaculture. In this review, we endeavored to accommodate the LAMP methodology with its different recent improvements and an overview of its application for the detection of aquaculture-associated pathogens.

  8. Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum

    PubMed Central

    Duan, Ya Bing; Yang, Ying; Wang, Jian Xin; Liu, Cong Chao; He, Ling Ling; Zhou, Ming Guo

    2015-01-01

    Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60–64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods. PMID:26606972

  9. Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.

    PubMed

    Duan, Ya Bing; Yang, Ying; Wang, Jian Xin; Liu, Cong Chao; He, Ling Ling; Zhou, Ming Guo

    2015-01-01

    Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods. PMID:26606972

  10. Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

    PubMed

    Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

    2014-02-01

    A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection.

  11. Development of Mitochondrial Loop-Mediated Isothermal Amplification for Detection of the Small Liver Fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes)

    PubMed Central

    Nguyen, Nga Thi Bich; Truong, Nam Hai; De, Nguyen Van

    2012-01-01

    Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1c+F2), BIP(B1c+B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly specific assay (mito-OvLAMP) to distinguish DNA of O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and other trematodes (Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was applied using F3/B3 primer pairs to verify the specificity of the primers for O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of SYBR green I to the product in sunlight or under UV light. Only DNA from O. viverrini yielded amplification products by LAMP (and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10−4 ng DNA), indicating that this assay is 10 to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where O. viverrini is endemic, which can lead to more effective control of opisthorchiasis and trematodiasis. PMID:22322346

  12. Development of a highly sensitive loop-mediated isothermal amplification (LAMP) method for the detection of Loa loa.

    PubMed

    Fernández-Soto, Pedro; Mvoulouga, Prosper Obolo; Akue, Jean Paul; Abán, Julio López; Santiago, Belén Vicente; Sánchez, Miguel Cordero; Muro, Antonio

    2014-01-01

    The filarial parasite Loa loa, the causative agent of loiasis, is endemic in Central and Western Africa infecting 3-13 million people. L. loa has been associated with fatal encephalopathic reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. In endemic areas, the only diagnostic method routinely used is the microscopic examination of mid-day blood samples by thick blood film. Improved methods for detection of L. loa are needed in endemic regions with limited resources, where delayed diagnosis results in high mortality. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid, inexpensive, molecular diagnosis of loiasis. Primers for LAMP were designed from a species-specific repetitive DNA sequence from L. loa retrieved from GenBank. Genomic DNA of a L. loa adult worm was used to optimize the LAMP conditions using a thermocycler or a conventional heating block. Amplification of DNA in the LAMP mixture was visually inspected for turbidity as well as addition of fluorescent dye. LAMP specificity was evaluated using DNA from other parasites; sensitivity was evaluated using DNA from L. loa 10-fold serially diluted. Simulated human blood samples spiked with DNA from L. loa were also tested for sensitivity. Upon addition of fluorescent dye, all positive reactions turned green while the negative controls remained orange under ambient light. After electrophoresis on agarose gels, a ladder of multiple bands of different sizes could be observed in positive samples. The detection limit of the assay was found to be as little as 0.5 ag of L. loa genomic DNA when using a heating block. We have designed, for the first time, a highly sensitive LAMP assay for the detection of L. loa which is potentially adaptable for field diagnosis and disease surveillance in loiasis-endemic areas. PMID:24722638

  13. Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes).

    PubMed

    Le, Thanh Hoa; Nguyen, Nga Thi Bich; Truong, Nam Hai; De, Nguyen Van

    2012-04-01

    Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1c+F2), BIP(B1c+B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly specific assay (mito-OvLAMP) to distinguish DNA of O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and other trematodes (Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was applied using F3/B3 primer pairs to verify the specificity of the primers for O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of SYBR green I to the product in sunlight or under UV light. Only DNA from O. viverrini yielded amplification products by LAMP (and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10(-4) ng DNA), indicating that this assay is 10 to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where O. viverrini is endemic, which can lead to more effective control of opisthorchiasis and trematodiasis. PMID:22322346

  14. Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick

    PubMed Central

    2014-01-01

    Background Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions Our results indicate that the methodology here reported constitutes a step forward in the development

  15. Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.

    PubMed

    Nair, Smita; Manimekalai, Ramaswamy; Ganga Raj, Palliyath; Hegde, Vinayaka

    2016-07-01

    The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings. PMID:27263003

  16. Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.

    PubMed

    Nair, Smita; Manimekalai, Ramaswamy; Ganga Raj, Palliyath; Hegde, Vinayaka

    2016-07-01

    The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings.

  17. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants.

    PubMed

    Niessen, Ludwig; Luo, Jie; Denschlag, Carla; Vogel, Rudi F

    2013-12-01

    Bacterial pathogens and toxicants, parasites as well as mycotoxin producing fungi are the major biotic factors influencing the safety of food. Moreover, viral infections and prions may be present as quasi biotic challenging factors. A vast array of culture dependent analytical methods and protocols for food safety testing has been developed during the past decades. Presently, protocols involving molecular biological techniques such as PCR-based nucleic acid amplification and hybridization have become available for many of the known pathogens with their major advantages being rapidness, high sensitivity and specificity. However, this type of assays is still quite labor- and cost intensive and mostly cannot be operated directly in the field. Recently, loop-mediated isothermal amplification (LAMP) of DNA has emerged as an alternative to the use of PCR-based methods not only in food safety testing but also in a wide array of application. Its advantages over PCR-based techniques are even shorter reaction time, no need for specific equipment, high sensitivity and specificity as well as comparably low susceptibility to inhibitors present in sample materials which enables detection of the pathogens in sample materials even without time consuming sample preparation. The present article presents a critical review of the application of LAMP-based methods and their usefulness in detecting and identifying food borne bacterial pathogens and toxicants as well as mycotoxin producing food borne fungi as compared to other methods. Moreover does it elaborate on new developments in the design and automation of LAMP-based assays and their implications for the future developments of food testing.

  18. Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes).

    PubMed

    Le, Thanh Hoa; Nguyen, Nga Thi Bich; Truong, Nam Hai; De, Nguyen Van

    2012-04-01

    Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1c+F2), BIP(B1c+B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly specific assay (mito-OvLAMP) to distinguish DNA of O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and other trematodes (Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was applied using F3/B3 primer pairs to verify the specificity of the primers for O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of SYBR green I to the product in sunlight or under UV light. Only DNA from O. viverrini yielded amplification products by LAMP (and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10(-4) ng DNA), indicating that this assay is 10 to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where O. viverrini is endemic, which can lead to more effective control of opisthorchiasis and trematodiasis.

  19. Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

    PubMed Central

    2014-01-01

    Background The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections. Methods Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region. Results The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR. Conclusions These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections. PMID:25103205

  20. Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Loa loa

    PubMed Central

    Fernández-Soto, Pedro; Mvoulouga, Prosper Obolo; Akue, Jean Paul; Abán, Julio López; Santiago, Belén Vicente; Sánchez, Miguel Cordero; Muro, Antonio

    2014-01-01

    The filarial parasite Loa loa, the causative agent of loiasis, is endemic in Central and Western Africa infecting 3–13 million people. L. loa has been associated with fatal encephalopathic reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. In endemic areas, the only diagnostic method routinely used is the microscopic examination of mid-day blood samples by thick blood film. Improved methods for detection of L. loa are needed in endemic regions with limited resources, where delayed diagnosis results in high mortality. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid, inexpensive, molecular diagnosis of loiasis. Primers for LAMP were designed from a species-specific repetitive DNA sequence from L. loa retrieved from GenBank. Genomic DNA of a L. loa adult worm was used to optimize the LAMP conditions using a thermocycler or a conventional heating block. Amplification of DNA in the LAMP mixture was visually inspected for turbidity as well as addition of fluorescent dye. LAMP specificity was evaluated using DNA from other parasites; sensitivity was evaluated using DNA from L. loa 10-fold serially diluted. Simulated human blood samples spiked with DNA from L. loa were also tested for sensitivity. Upon addition of fluorescent dye, all positive reactions turned green while the negative controls remained orange under ambient light. After electrophoresis on agarose gels, a ladder of multiple bands of different sizes could be observed in positive samples. The detection limit of the assay was found to be as little as 0.5 ag of L. loa genomic DNA when using a heating block. We have designed, for the first time, a highly sensitive LAMP assay for the detection of L. loa which is potentially adaptable for field diagnosis and disease surveillance in loiasis-endemic areas. PMID:24722638

  1. Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B.

    PubMed

    Cao, Cuiming; Zhang, Fang; Ji, Mingyu; Pei, Fengyan; Fan, Xiujie; Shen, Hong; Wang, Qingxi; Yang, Weihua; Wang, Yunshan

    2016-07-01

    We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 °C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng/μl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng/μl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries. PMID:27045360

  2. Sensitive and Rapid Detection of the New Delhi Metallo-Beta-Lactamase Gene by Loop-Mediated Isothermal Amplification

    PubMed Central

    Liu, Wei; Zou, Dayang; Li, Yan; Wang, Xuesong; He, Xiang; Wei, Xiao; Shao, Changlin; Li, Xuelian; Shang, Wei; Yu, Kaitao; Liu, Dawei; Li, Yunmei; Guo, Jing; Yin, Zhitao

    2012-01-01

    New Delhi metallo-β-lactamase 1 (NDM-1), which is associated with resistance to carbapenem, was first reported in 2008. A sensitive and rapid molecular assay to detect the plasmid blaNDM-1 in clinical isolates is needed to control its spread. We describe a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of blaNDM-1 from pure culture and sputum, urine, and fecal samples. Eight sets of primers were designed to recognize six or eight distinct sequences on target blaNDM-1, and one set was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for blaNDM-1 detection were determined. The sensitivity of the LAMP assay for blaNDM-1 detection in sputum, urine, and fecal samples was also tested. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 70 min at an isothermal temperature of 65°C. The sensitivity of LAMP, with a detection limit of 10.70 pg/μl DNA, was 100-fold greater than that of PCR. Thirteen infection bacterial strains without blaNDM-1 were selected for testing of specificity, and the results of the amplification were negative, which showed that the primers had good levels of specificity. The LAMP method reported here is demonstrated to be a potentially valuable means for the detection of blaNDM-1 and rapid clinical diagnosis, being fast, simple, and low in cost. PMID:22357496

  3. Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

    PubMed

    Tsai, Shinn Shyong; Chang, Yeng Ling; Huang, Yen Li; Liu, Hung Jen; Ke, Guan Ming; Chiou, Chwei Jang; Hsieh, Yao Ching; Chang, Tsung Chou; Cheng, Li Ting; Chuang, Kuo Pin

    2014-05-01

    There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity. PMID:24193953

  4. Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

    PubMed

    Tsai, Shinn Shyong; Chang, Yeng Ling; Huang, Yen Li; Liu, Hung Jen; Ke, Guan Ming; Chiou, Chwei Jang; Hsieh, Yao Ching; Chang, Tsung Chou; Cheng, Li Ting; Chuang, Kuo Pin

    2014-05-01

    There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity.

  5. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples.

    PubMed

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; van Doorn, Joop; Pham, Khanh; Cambra, Miguel A; Berruete, Isabel M; Pothier, Joël F; Duffy, Brion; Olmos, Antonio; López, María M

    2015-05-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparation were analyzed. Kappa coefficient, sensitivity, specificity, likelihood ratios and post-test probability of detection were estimated to manage the risk associated with the use of the two methods.

  6. Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity.

    PubMed

    Mitsunaga, Shigeki; Shimizu, Sayoko; Okudaira, Yuko; Oka, Akira; Tanaka, Masafumi; Kimura, Minoru; Kulski, Jerzy K; Inoue, Ituro; Inoko, Hidetoshi

    2013-06-01

    Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.

  7. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

    PubMed

    Poole, Catherine B; Ettwiller, Laurence; Tanner, Nathan A; Evans, Thomas C; Wanji, Samuel; Carlow, Clotilde K S

    2015-01-01

    Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25-30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal. PMID:26414073

  8. Surveillance for Western equine encephalitis St. Louis encephalitis and West Nile viruses using reverse transcription loop-mediated isothermal amplification

    DOE PAGES

    Meagher, Robert J.; Ball, Cameron Scott; Langevin, Stanley A.; Fang, Ying; Wheeler, Sarah S.; Coffey, Lark L.

    2016-01-25

    In this study, collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized publicmore » health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.« less

  9. Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan.

    PubMed

    Arai, Sakura; Tohya, Mari; Yamada, Ryoko; Osawa, Ro; Nomoto, Ryohei; Kawamura, Yoshiaki; Sekizaki, Tsutomu

    2015-09-01

    We here developed a novel loop-mediated isothermal amplification (LAMP) method to detect Streptococcus suis in raw pork meat. This method, designated LAMPSS, targeted the recombination/repair protein (recN) gene of S. suis and detected all serotypes of S. suis, except those taxonomically removed from authentic S. suis, i.e., serotypes 20, 22, 26, 32, 33, and 34. The specificity of LAMPSS was confirmed and its detection limit was 5.4cfu/reaction. Among the 966 raw pork meat samples examined, including sliced pork, minced pork, and the liver, tongue, heart, and small intestine, 255 samples tested positive with LAMPSS. The rate of contamination was higher in the organs than in pork. No significant difference was observed in the total bacterial count between LAMPSS-positive and -negative samples. The number of shops that provided LAMPSS-positive pork was slightly higher in those that sold swine organs and pork than in those that sold only pork, suggesting that cross contamination occurred from the organs to pork. Among the 255 which tested positive for LAMPSS, only 47 samples tested positive for the previously described LAMP specific for S. suis serotype 2. Two isolates of S. suis serotype 2, belonging to sequence type 28, which is potentially hazardous to humans, as well as those of some other serotypes were obtained from 19 out of 47 samples by combining LAMP with a replica plating method. These results suggest that LAMPSS will be a useful tool for the surveillance of raw pork meat in the retail market.

  10. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

    PubMed

    Poole, Catherine B; Ettwiller, Laurence; Tanner, Nathan A; Evans, Thomas C; Wanji, Samuel; Carlow, Clotilde K S

    2015-01-01

    Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25-30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.

  11. Performance of reversed transcription loop-mediated isothermal amplification technique detecting EV71: a systematic review with meta-analysis.

    PubMed

    Lei, Xiaoying; Wen, Hongling; Zhao, Li; Yu, Xuejie

    2014-04-01

    Human enterovirus 71 (EV71) is the major etiological agent of hand, foot and mouth disease (HFMD), which is a common infectious disease in young children. Studies in the past have shown that reversed transcription loop-mediated isothermal amplification (RT-LAMP) was a rapid approach for the detection of EV71 in HFMD. This meta-analysis study is to evaluate the diagnostic role of RT-LAMP in detecting EV71 infection. A comprehensive literature research of PubMed, Embase, Wan Fang Data, and Chinese National Knowledge Infrastructure databases was conducted on articles aiming at the diagnostic performance of RT-LAMP in EV71 detection published before February 10, 2014. Data from selected studies were pooled to yield the summary sensitivity, specificity, positive and negative likelihood ratio (PLR, NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (SROC) curve by using STATA VERSION 12.0 software. Ten studies including a total of 907 clinical samples were of high quality in this meta-analysis. Overall, the pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve was 0.99 (0.97, 1.00), 0.97 (0.94, 1.00), 5.90 (95% CI: 3.90-8.94), 0.20 (95% CI: 0.14-0.29), and 1.00 (95% CI: 0.99-1.00), respectively. The univariate analysis of potential variables showed some changes in the diagnostic performance, but none of the differences reached statistical significance. Despite inter-study variability, the test performance of RT-LAMP was consistent with real-time RT-PCR in detecting EV71. This meta-analysis suggests that RT-LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting EV71. PMID:24815384

  12. Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

    PubMed Central

    Zhou, Yitian; Theodoro, Raquel C.; Abrams, Bethany; Balajee, S. Arunmozhi; Litvintseva, Anastasia P.

    2014-01-01

    Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n = 6) collected from HIV-positive individuals with culture- and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n = 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was ≤6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted. PMID:24478477

  13. Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Malaria Infections in an Area of Endemicity in Thailand

    PubMed Central

    Sattabongkot, Jetsumon; Tsuboi, Takafumi; Han, Eun-Taek; Bantuchai, Sirasate

    2014-01-01

    The loop-mediated isothermal amplification (LAMP) method, developed by our group for diagnosis of four human malaria parasites, was evaluated on a large scale at a remote clinic in Thailand where malaria is endemic. A total of 899 febrile patients were analyzed in this study. LAMP was first evaluated in 219 patients, and the result was compared to those of two histidine-rich protein (HRP)-2 rapid diagnostic tests (RDTs) and microscopy as a gold standard. LAMP DNA extraction was conducted by a simple boiling method, and the test results were assessed visually. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 95.7%, 100%, 100%, and 98%, respectively, for LAMP and 98.6%, 98%, 95.8%, and 99.3%, respectively, for RDTs. Since RDT-positive results were based on one out of two RDTs, the sensitivity of RDTs was slightly higher than that of LAMP. However, LAMP tended to be more specific than RDTs. LAMP next was evaluated in 680 patients, and the result was compared to that of microscopy as a gold standard. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of LAMP were 88.9%, 96.9%, 92.2%, 95.5%, and 94.6%, respectively. Nested PCR was used to confirm the discrepant results. Malaria LAMP in a remote clinic in Thailand achieved an acceptable result, indicating that LAMP malaria diagnosis is feasible in a field setting with limited technical resources. Additionally, the rapid boiling method for extracting DNA from dried blood spots proved to be simple, fast, and suitable for use in the field. PMID:24574279

  14. Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification

    PubMed Central

    Wheeler, Sarah S.; Ball, Cameron S.; Langevin, Stanley A.; Fang, Ying; Coffey, Lark L.; Meagher, Robert J.

    2016-01-01

    Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance. PMID:26807734

  15. Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification.

    PubMed

    Wheeler, Sarah S; Ball, Cameron S; Langevin, Stanley A; Fang, Ying; Coffey, Lark L; Meagher, Robert J

    2016-01-01

    Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3' untranslated region (3'-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.

  16. Performance of reversed transcription loop-mediated isothermal amplification technique detecting EV71: a systematic review with meta-analysis.

    PubMed

    Lei, Xiaoying; Wen, Hongling; Zhao, Li; Yu, Xuejie

    2014-04-01

    Human enterovirus 71 (EV71) is the major etiological agent of hand, foot and mouth disease (HFMD), which is a common infectious disease in young children. Studies in the past have shown that reversed transcription loop-mediated isothermal amplification (RT-LAMP) was a rapid approach for the detection of EV71 in HFMD. This meta-analysis study is to evaluate the diagnostic role of RT-LAMP in detecting EV71 infection. A comprehensive literature research of PubMed, Embase, Wan Fang Data, and Chinese National Knowledge Infrastructure databases was conducted on articles aiming at the diagnostic performance of RT-LAMP in EV71 detection published before February 10, 2014. Data from selected studies were pooled to yield the summary sensitivity, specificity, positive and negative likelihood ratio (PLR, NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (SROC) curve by using STATA VERSION 12.0 software. Ten studies including a total of 907 clinical samples were of high quality in this meta-analysis. Overall, the pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve was 0.99 (0.97, 1.00), 0.97 (0.94, 1.00), 5.90 (95% CI: 3.90-8.94), 0.20 (95% CI: 0.14-0.29), and 1.00 (95% CI: 0.99-1.00), respectively. The univariate analysis of potential variables showed some changes in the diagnostic performance, but none of the differences reached statistical significance. Despite inter-study variability, the test performance of RT-LAMP was consistent with real-time RT-PCR in detecting EV71. This meta-analysis suggests that RT-LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting EV71.

  17. Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.

    PubMed

    Yang, Qianru; Wang, Fei; Jones, Kelly L; Meng, Jianghong; Prinyawiwatkul, Witoon; Ge, Beilei

    2015-04-01

    Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.

  18. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    PubMed Central

    Poole, Catherine B.; Ettwiller, Laurence; Tanner, Nathan A.; Evans, Thomas C.; Wanji, Samuel; Carlow, Clotilde K. S.

    2015-01-01

    Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal. PMID:26414073

  19. Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye.

    PubMed

    Almasi, Mohammad Amin; Erfan Manesh, Maryam; Jafary, Hossein; Dehabadi, Seyed Mohammad Hosseini

    2013-09-01

    The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock. PLRV RNA was extracted from overall 80 infected naturally potato leaves. A set of six novel primers for the LAMP reaction was designed according to the highly conserved sequence of the viral coat protein (CP) gene. LAMP was carried out under isothermal conditions, applying the Bst DNA polymerase enzyme; the LAMP products were detected visually using the GeneFinder™ florescence dye. A positive result using the GeneFinder™ dye was a color change from the original orange to green. Results confirmed LAMP with GeneFinder™ provides a rapid and safe assay for detection of PLRV. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostic methods in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations.

  20. Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan.

    PubMed

    Arai, Sakura; Tohya, Mari; Yamada, Ryoko; Osawa, Ro; Nomoto, Ryohei; Kawamura, Yoshiaki; Sekizaki, Tsutomu

    2015-09-01

    We here developed a novel loop-mediated isothermal amplification (LAMP) method to detect Streptococcus suis in raw pork meat. This method, designated LAMPSS, targeted the recombination/repair protein (recN) gene of S. suis and detected all serotypes of S. suis, except those taxonomically removed from authentic S. suis, i.e., serotypes 20, 22, 26, 32, 33, and 34. The specificity of LAMPSS was confirmed and its detection limit was 5.4cfu/reaction. Among the 966 raw pork meat samples examined, including sliced pork, minced pork, and the liver, tongue, heart, and small intestine, 255 samples tested positive with LAMPSS. The rate of contamination was higher in the organs than in pork. No significant difference was observed in the total bacterial count between LAMPSS-positive and -negative samples. The number of shops that provided LAMPSS-positive pork was slightly higher in those that sold swine organs and pork than in those that sold only pork, suggesting that cross contamination occurred from the organs to pork. Among the 255 which tested positive for LAMPSS, only 47 samples tested positive for the previously described LAMP specific for S. suis serotype 2. Two isolates of S. suis serotype 2, belonging to sequence type 28, which is potentially hazardous to humans, as well as those of some other serotypes were obtained from 19 out of 47 samples by combining LAMP with a replica plating method. These results suggest that LAMPSS will be a useful tool for the surveillance of raw pork meat in the retail market. PMID:26043307

  1. Development and evaluation of loop-mediated isothermal amplification assay for detection of Crimean Congo hemorrhagic fever virus in Sudan.

    PubMed

    Osman, Hana A M; Eltom, Kamal H; Musa, Nasreen O; Bilal, Nasreldin M; Elbashir, Mustafa I; Aradaib, Imadeldin E

    2013-06-01

    Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) activity has been detected in Kordufan region of the Sudan in 2008 with high case-fatality rates in villages and rural hospitals in the region. Therefore, in the present study, a reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed and compared to nested RT-PCR for rapid detection of CCHFV targeting the small (S) RNA segment. A set of RT-LAMP primers, designed from a highly conserved region of the S segment of the viral genome, was employed to identify all the Sudanese CCHFV strains. The sensitivity studies indicated that the RT-LAMP detected 10fg of CCHFV RNA as determined by naked eye turbidity read out, which is more likely the way it would be read in a resource-poor setting. This level of sensitivity is good enough to detect most acute cases. Using agarose gel electrophoresis, the RT-LAMP assay detected as little as 0.1fg of viral RNA (equivalent to 50 viral particle). There was 100% agreement between results of the RT-LAMP and the nested PCR when testing 10-fold serial dilution of CCHFV RNA. The specificity studies indicated that there was no cross-reactivity with other related hemorrhagic fever viruses circulating in Sudan including, Rift Valley fever virus (RVFV), Dengue fever virus, and yellow fever virus. The RT-LAMP was performed under isothermal conditions at 63°C and no special apparatus was needed, which rendered the assay more economical and practical than real-time PCR in such developing countries, like Sudan. In addition, the RT-LAMP provides a valuable tool for rapid detection and differentiation of CCHFV during an outbreak of the disease in remote areas and in rural hospitals with resource-poor settings.

  2. Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop-mediated isothermal amplification assay.

    PubMed

    Nagai, Yuhki; Iwade, Yoshito; Nakano, Manabu; Akachi, Shigehiro; Kobayashi, Takashi; Nishinaka, Takamichi

    2016-07-01

    Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug-resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing-specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time-consuming and technically demanding. In the present study, a loop-mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain-identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c-targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207-PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c-multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains. PMID:27213686

  3. Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.

    PubMed

    Prusty, Bikash R; Chaudhuri, Pallab; Chaturvedi, V K; Saini, Mohini; Mishra, B P; Gupta, Praveen K

    2016-06-01

    Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen. PMID:27570305

  4. Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification.

    PubMed

    Wheeler, Sarah S; Ball, Cameron S; Langevin, Stanley A; Fang, Ying; Coffey, Lark L; Meagher, Robert J

    2016-01-01

    Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3' untranslated region (3'-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance. PMID:26807734

  5. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    PubMed

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  6. [Investigation on Toxoplasma gondii by polymerase chain reaction and loop-mediated isothermal amplification in water samples from Giresun, Turkey].

    PubMed

    Demirel, Elif; Kolören, Zeynep; Karaman, Ulkü; Ayaz, Emine

    2014-10-01

    Toxoplasmosis is generally asymptomatic in immunocompetent subjects, however serious manifestations of the disease may develop in immunocompromised patients and in pregnant women. The mean Toxoplasma gondii seroprevalence which is approximately 40% in Turkish population, indicates the high risk for the development of acute toxoplasmosis in those cases. One of the transmission ways of T.gondii is the consumption of contaminated water. The aim of this study was to detect the presence of T.gondii in the environmental and drinking water samples by using standard polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods. A total of 96 water samples, of them 76 were environmental water samples collected from creeks in Giresun province center and its various districts (Piraziz, Bulancak, Keşap, Espiye) and 20 from drinking water samples, were included in the study. The samples were precipitated by the aluminium sulfate method and DNAs were isolated from the pellets formed with the sucrose gradient method. PCR and LAMP were applied to the isolated DNAs, by the use of primers F3 and B3 specific to B1 gene of the parasite. In our study, T.gondii was detected in none of the drinking water samples by PCR and LAMP, however T.gondii DNAs were positive in 13.2% (10/76) of the environmental water samples. Both of the methods yielded the same results in those samples. The stations that were positive for T.gondii were Aksu creek in Giresun province center; Gelivera creek in Espiye; Yolağzı, Keşap, Keşap Login Bridge creeks in Keşap; Bulancak, Karadere and İncivez creeks in Bulancak; Piraziz and Çayırağzı creeks in Piraziz counties. Resistance of T.gondii to chlorination and the inadequacy of the filtration processes, create a serious threat among people in contact with rivers, sea and drinking waters particularly in areas with high humidity. As far as the national literature was considered, no report about the water-borne T

  7. Diagnostic accuracy of loop-mediated isothermal amplification in detection of Clostridium difficile in stool samples: a meta-analysis

    PubMed Central

    Wei, Chen; Yang-Ming, Li; Shan, Luo; Yi-Ming, Zhong

    2015-01-01

    Introduction Clostridium difficile infection (CDI) remains a diagnostic challenge for clinicians. More recently, loop-mediated isothermal amplification (LAMP) has become readily available for the diagnosis of CDI, and many studies have investigated the usefulness of LAMP for rapid and accurate diagnosis of CDI. However, the overall diagnostic accuracy of LAMP for CDI remains unclear. In this meta-analysis, our aim was to establish the overall diagnostic accuracy of LAMP in detection of Clostridium difficile (CD) in stool samples. Material and methods A search was done in PubMed, MEDLINE, EMBASE and Cochrane Library databases up to February 2014 to identify published studies that evaluated the diagnostic role of LAMP for CD. Methodological quality was assessed according to the quality assessment for studies of diagnostic accuracy (QUADAS) instrument. The sensitivities (SEN), specificities (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) were pooled statistically using random effects models. Statistical analysis was performed by employing Meta-Disc 1.4 software. Summary receiver operating characteristic (SROC) curves were used to summarize overall test performance. Funnel plots were used to test the potential publication bias. Result A total of 9 studies met inclusion criteria for the present meta-analysis. The pooled SEN and SPE for diagnosing CD were 0.93 (95% CI: 0.91–0.95) and 0.98 (95% CI: 0.98–0.99), respectively. The PLR was 47.72 (95% CI: 15.10–150.82), NLR was 0.07 (95% CI: 0.04–0.14) and DOR was 745.19 (95% CI: 229.30−2421.72). The area under the ROC was 0.98. Meta-regression indicated that the total number of samples was a source of heterogeneity for LAMP in detection of CD. The funnel plots suggested no publication bias. Conclusions The LAMP meets the minimum desirable characteristics of a diagnostic test of SEN, SPE and other measures of accuracy in the diagnosis of CD, and it is suitable

  8. Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

    PubMed Central

    Beissner, Marcus; Phillips, Richard Odame; Battke, Florian; Bauer, Malkin; Badziklou, Kossi; Sarfo, Fred Stephen; Maman, Issaka; Rhomberg, Agata; Piten, Ebekalisai; Frimpong, Michael; Huber, Kristina Lydia; Symank, Dominik; Jansson, Moritz; Wiedemann, Franz Xaver; Banla Kere, Abiba; Herbinger, Karl-Heinz; Löscher, Thomas; Bretzel, Gisela

    2015-01-01

    Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold

  9. [Investigation on Toxoplasma gondii by polymerase chain reaction and loop-mediated isothermal amplification in water samples from Giresun, Turkey].

    PubMed

    Demirel, Elif; Kolören, Zeynep; Karaman, Ulkü; Ayaz, Emine

    2014-10-01

    Toxoplasmosis is generally asymptomatic in immunocompetent subjects, however serious manifestations of the disease may develop in immunocompromised patients and in pregnant women. The mean Toxoplasma gondii seroprevalence which is approximately 40% in Turkish population, indicates the high risk for the development of acute toxoplasmosis in those cases. One of the transmission ways of T.gondii is the consumption of contaminated water. The aim of this study was to detect the presence of T.gondii in the environmental and drinking water samples by using standard polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods. A total of 96 water samples, of them 76 were environmental water samples collected from creeks in Giresun province center and its various districts (Piraziz, Bulancak, Keşap, Espiye) and 20 from drinking water samples, were included in the study. The samples were precipitated by the aluminium sulfate method and DNAs were isolated from the pellets formed with the sucrose gradient method. PCR and LAMP were applied to the isolated DNAs, by the use of primers F3 and B3 specific to B1 gene of the parasite. In our study, T.gondii was detected in none of the drinking water samples by PCR and LAMP, however T.gondii DNAs were positive in 13.2% (10/76) of the environmental water samples. Both of the methods yielded the same results in those samples. The stations that were positive for T.gondii were Aksu creek in Giresun province center; Gelivera creek in Espiye; Yolağzı, Keşap, Keşap Login Bridge creeks in Keşap; Bulancak, Karadere and İncivez creeks in Bulancak; Piraziz and Çayırağzı creeks in Piraziz counties. Resistance of T.gondii to chlorination and the inadequacy of the filtration processes, create a serious threat among people in contact with rivers, sea and drinking waters particularly in areas with high humidity. As far as the national literature was considered, no report about the water-borne T

  10. Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

    PubMed Central

    González, Iveth J.; Polley, Spencer D.; Bell, David; Shakely, Delér; Msellem, Mwinyi I.; Björkman, Anders; Mårtensson, Andreas

    2014-01-01

    Background Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Methods and Findings Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups. Conclusion Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low

  11. [Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

    PubMed

    Lin, Feng; Liu, Li; Hao, Gui-Jie; Cao, Zheng; Sheng, Peng-Cheng; Wu, Ying-Lei; Shen, Jin-Yu

    2014-09-01

    White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection. PMID:25562958

  12. [Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

    PubMed

    Lin, Feng; Liu, Li; Hao, Gui-Jie; Cao, Zheng; Sheng, Peng-Cheng; Wu, Ying-Lei; Shen, Jin-Yu

    2014-09-01

    White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection.

  13. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    PubMed

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection. PMID:27400833

  14. Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

    PubMed Central

    Kim, Eun-Mi; Jeon, Hyo-Sung; Kim, Ji-Jung; Shin, Yeun-Kyung; Lee, Youn-Jeong; Yeo, Sang-Geon

    2016-01-01

    Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples. PMID:26726027

  15. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis.

    PubMed

    Balne, P K; Basu, S; Rath, S; Barik, M R; Sharma, S

    2015-01-01

    This study is a comparative evaluation (Chi-square test) of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP), real-time polymerase chain reaction (PCR) and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8%) was higher (not significant, P value 0.2) than conventional PCR (57.6%) and lower than real-time PCR (90.9%). Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20) by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.

  16. Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.

    PubMed

    Mansour, Shimaa M G; Ali, Haytham; Chase, Christopher C L; Cepica, Arnost

    2015-12-01

    Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities.

  17. Loop region-specific oligonucleotide probes for loop-mediated isothermal amplification-enzyme-linked immunosorbent assay truly minimize the instrument needed for detection process.

    PubMed

    Ravan, Hadi; Yazdanparast, Razieh

    2013-08-15

    Enteric fever represents a significant public health burden in less-developed countries. Therefore, there is a great need for developing an improved diagnostic tool adapted to the demands of poor-resource clinical laboratories in those countries. The current study has developed a reliable loop-mediated isothermal amplification (LAMP)-enzyme-linked immunosorbent assay (ELISA) for diagnosis of enteric fever with a minimal equipment dependency. The LAMP-ELISA assay involves direct incorporation of a labeled nucleotide into amplicons during the amplification of the SPA3440 gene, their hybridization to the unique tagged oligonucleotide probes during the LAMP reaction, and finally detection of labeled LAMP amplicons by immunoassay technology. Because the designed oligonucleotide probes target the single-stranded DNA segment within the LAMP amplicons, the probe hybridization stage is performed simultaneously with the amplification process. This novel probe design strategy allows both the amplification and hybridization stages to be performed simultaneously and isothermally in a water bath. Among the bacteria tested, positive results were observed only with enteric fever causative bacteria. The LAMP-ELISA assay was successfully applied to artificially contaminated blood samples with a detection limit of 10 colony-forming units (CFU)/ml, which was 100 times more sensitive than polymerase chain reaction (PCR) and turbidity assessment-based conventional LAMP methods. The new assay is considered to be an effective method for diagnosis of enteric fever.

  18. Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

    PubMed

    Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Plutzer, Judit; Noack, Michael J; Mahmoudi, Mohammad Reza; Karanis, Panagiotis

    2016-06-01

    Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters.

  19. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

    PubMed

    Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

    2015-03-01

    Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs. PMID:25582179

  20. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-05-26

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies.

  1. Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection.

    PubMed

    Nakauchi, Mina; Takayama, Ikuyo; Takahashi, Hitoshi; Tashiro, Masato; Kageyama, Tsutomu

    2014-08-01

    A genetic diagnosis system for detecting avian influenza A (H7N9) virus infection using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology was developed. The RT-LAMP assay showed no cross-reactivity with seasonal influenza A (H3N2 and H1N1pdm09) or influenza B viruses circulating in humans or with avian influenza A (H5N1) viruses. The sensitivity of the RT-LAMP assay was 42.47 copies/reaction. Considering the high specificity and sensitivity of the assay for detecting the avian influenza A (H7N9) virus and that the reaction was completed within 30 min, the RT-LAMP assay developed in this study is a promising rapid diagnostic tool for avian influenza A (H7N9) virus infection.

  2. Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

    2014-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.

  3. Detection of early and single infections of Schistosoma japonicum in the intermediate host snail, Oncomelania hupensis, by PCR and loop-mediated isothermal amplification (LAMP) assay.

    PubMed

    Kumagai, Takashi; Furushima-Shimogawara, Rieko; Ohmae, Hiroshi; Wang, Tian-Ping; Lu, Shaohong; Chen, Rui; Wen, Liyong; Ohta, Nobuo

    2010-09-01

    Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places. PMID:20810818

  4. Detection and differentiation of Japanese encephalitis virus genotype I and genotype III by reverse transcription loop-mediated isothermal amplification combined with restriction fragment length polymorphism.

    PubMed

    Zhang, Liang; Cao, Sanjie; Wu, Rui; Zhu, Shuquan; Liu, Hanyang; Yuan, Lei; Shi, Shuangyan; Zhang, Dan; Huang, Xiaobo; Wen, Xintian; Wen, Yiping; Yan, Qigui; Huang, Yong; Ma, Xiaoping

    2015-04-01

    Japanese encephalitis (JE), which is a mosquito-borne arboviral infection, is the leading cause of viral encephalitis in Asian countries. The causative agent of JE is Japanese encephalitis virus (JEV), in which the predominant genotype has changed from genotype III (G III) to genotype I (G I). However, a method for the rapid differentiation between JEV G I and G III remains unavailable. This study aimed to establish a rapid JEV genotyping method using reverse transcription loop-mediated isothermal amplification (RT-LAMP). An Spe I site, which was located in the target sequence (C gene) of JEV G III strains but not in JEV G I strains, was selected as the RT-LAMP target. After testing 64 specimens, results showed that RT-LAMP can detect and differentiate JEV G I and G III specifically. Thus, a novel RT-LAMP system for the rapid detection and differentiation of JEV G I and G III was developed successfully.

  5. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

    PubMed

    Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

    2015-03-01

    Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs.

  6. Loop-mediated isothermal amplification (RT-LAMP): a new approach for the detection of foot-and-mouth disease virus and its sero-types in Pakistan.

    PubMed

    Farooq, U; Latif, A; Irshad, H; Ullah, A; Zahur, A B; Naeem, K; Khan, S U H; Ahmed, Z; Rodriguez, L L; Smoliga, G

    2015-01-01

    Successful disease management requires a rapid and sensitive diagnosis method that can recognize early infection even before the manifestation of its clinical signs. The only available field diagnostic tests for foot-and-mouth disease (FMD) are lateral flow devices, commonly known as chromatographic strips. Low sensitivity and inability to detect FMD virus (FMDV) at the serotype level are limitations of lateral flow devices. Therefore, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was standardized using universal and sero-type specific genes in a single tube. This test does not require sophisticated equipment and can detect FMDV at serotype level in about 60 min. In addition, the sensitivity and specificity of this test is comparable to conventional reverse transcriptase PCR and real time PCR (rRT-PCR). PMID:27175198

  7. Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

    2014-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd. PMID:24631346

  8. Evaluation of Two Loop-mediated Isothermal Amplification Methods for the Detection of Salmonella Enteritidis and Listeria Monocytogenes in Artificially Contaminated Ready-to-Eat Fresh Produce

    PubMed Central

    Birmpa, Angeliki; Kalogeropoulos, Konstantinos; Kokkinos, Petros

    2015-01-01

    In the present study, the effectiveness of two loop-mediated isothermal amplification (LAMP) assays was evaluated. Samples of romaine lettuce, strawberries, cherry tomatoes, green onions and sour berries were inoculated with known dilutions (100-108 CFU/g of produce) of S. Enteritidis and L. monocytogenes. With LAMP, assay pathogens can be detected in less than 60 min. The limits of detection of S. Enteritidis and L. monocytogenes depended on the food sample tested and on the presence of enrichment step. After enrichment steps, all food samples were found positive even at low initial pathogen levels. The developed LAMP, assays, are expected to become a valuable, robust, innovative, powerful, cheap and fast monitoring tool, which can be extensively used for routine analysis, and screening of contaminated foods by the food industry and the Public Food Health Authorities. PMID:27800413

  9. Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease.

    PubMed

    Zhou, Shun; Gao, Zhi-Xin; Zhang, Min; Liu, Dan-Yang; Zhao, Xin-Peng; Liu, Yong

    2016-01-01

    A quadruplex loop-mediated isothermal amplification (LAMP) method was developed to detect four Vibrio species, including Vibrio ichthyoenteri, Vibrio parahaemolyticus, Vibrio scophthalmi, and Vibrio vulnificus, simultaneously. Four sets of species-specific primers were designed with different restriction sites contained in the inner primers. The quadruplex LAMP method could distinguish four Vibrio species via the subsequent restriction enzyme analysis. The sensitivity of the quadruplex LAMP method were 10(2)-10(3) times higher than the sensitivity of conventional PCR. V. scophthalmi, V. vulnificus, V. parahaemolyticus and V. ichthyoenteri could be detected in the different tissues of the infected fish by the quadruplex LAMP method simply and conveniently through using SYBR Green I to facilitate visual inspection of the LAMP products. The method we developed in this study could be a simple and convenient diagnostic tool for field detection of Vibrio infection in fish. PMID:27468405

  10. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    PubMed Central

    Wang, Deguo; Liu, Yanhong

    2015-01-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  11. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-06-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  12. An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step.

    PubMed

    Lee, Dohwan; Kim, Yong Tae; Lee, Jee Won; Kim, Do Hyun; Seo, Tae Seok

    2016-05-15

    We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h.

  13. A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples.

    PubMed

    Sun, Yi; Quyen, Than Linh; Hung, Tran Quang; Chin, Wai Hoe; Wolff, Anders; Bang, Dang Duong

    2015-04-21

    Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.

  14. Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

    PubMed Central

    Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.

    2015-01-01

    The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens. PMID:25856093

  15. Development of a loop-mediated isothermal amplification method for the rapid detection of the dioxin-degrading bacterium Ochrobactrum anthropi in soil.

    PubMed

    Chen, Hsi-Jien; Lee, Meng-Shiou; Lai, Jun-Yu; Lai, Guan-Hua

    2015-09-01

    In this study, loop-mediated isothermal amplification (LAMP) and real-time LAMP assays were developed to detect the dioxin-degrading bacterium Ochrobactrum anthropi strain BD-1 in soil. Four primers were designed to use ITS gene amplification for the strain O. anthropi BD-1. The real-time LAMP assay was found to accomplish the reaction by 1 pg of genomic DNA load when used for nucleic acid amplification. This assay was then applied to detect O. anthropi BD-1 in eight soil samples collected from a dioxin-contaminated site. The results demonstrated that these newly developed LAMP and real-time LAMP assays will not only be useful and efficient tools for detecting the target gene, but also be used as molecular tools for monitoring the growth of dioxin-degrading O. anthropi in the soil. This is the first report to demonstrate the use of LAMP assays to monitor the presence of O. anthropi in dioxin-contaminated soil. The application of this method should improve the biomonitoring of dioxin contamination.

  16. Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi.

    PubMed

    Hobo, Seiji; Niwa, Hidekazu; Oku, Kazuomi

    2012-03-01

    Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections.

  17. Visual Detection of West Nile Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Vertical Flow Visualization Strip

    PubMed Central

    Cao, Zengguo; Wang, Hualei; Wang, Lina; Li, Ling; Jin, Hongli; Xu, Changping; Feng, Na; Wang, Jianzhong; Li, Qian; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

    2016-01-01

    West Nile virus (WNV) causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF) was developed to detect the envelope (E) gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl of an WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubation of the amplification product on the visualization strip, and no cross-reaction with other closely related members of the Flavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV. The assay produced sensitivities of 101.5 TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field. PMID:27148234

  18. Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

    PubMed

    Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

    2015-06-01

    The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production. PMID:25771218

  19. Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates.

    PubMed

    Nakashima, Kei; Aoshima, Masahiro; Ohkuni, Yoshihiro; Hoshino, Eri; Hashimoto, Kohei; Otsuka, Yoshihito

    2014-12-01

    Loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We retrospectively evaluated 78 consecutive HIV-uninfected patients who underwent LAMP method for diagnosing Pneumocystis pneumonia (PCP). Diagnosis of PCP was made by the detection of Pneumocystis jirovecii (P. jirovecii) with positive LAMP or conventional staining (CS) (Grocott methenamine silver staining or Diff-Quick™) on the basis of compatible clinical symptoms and radiologic findings. Additionally, we reviewed HIV-uninfected immunocompromised patients who underwent subcontract PCR as a historical control. LAMP was positive in 10 (90.9%) of 11 positive-CS patients. Among 13 negative-CS patients with positive LAMP, 11 (84.6%) had PCP, and the remaining 2 were categorized as having P. jirovecii colonization. LDH levels in negative-CS PCP were higher than in positive-CS PCP (p = 0.026). (1 → 3)-β-D-glucan levels in negative-CS PCP were lower than in positive-CS PCP (p = 0.011). The interval from symptom onset to diagnosis as PCP in LAMP group (3.45 ± 1.77 days; n = 22) was shorter than in subcontract PCR group (6.90 ± 2.28 days; n = 10; p < 0.001). As for patients without PCP, duration of unnecessary PCP treatment in LAMP group (2; 2-3 days; n = 10) was shorter than in subcontract PCR group (7; 7-12.25 days; n = 6; p = 0.003). LAMP showed higher sensitivity (95.4%) and positive predictive value (91.3%) than subcontract PCR did. Pneumocystis LAMP method is a sensitive and cost-effective diagnostic method and is easy to administer in general hospitals. In-house LAMP method would realize early diagnosis of PCP, resulting in improving PCP prognosis and reducing unnecessary PCP-specific treatment.

  20. Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates.

    PubMed

    Nakashima, Kei; Aoshima, Masahiro; Ohkuni, Yoshihiro; Hoshino, Eri; Hashimoto, Kohei; Otsuka, Yoshihito

    2014-12-01

    Loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We retrospectively evaluated 78 consecutive HIV-uninfected patients who underwent LAMP method for diagnosing Pneumocystis pneumonia (PCP). Diagnosis of PCP was made by the detection of Pneumocystis jirovecii (P. jirovecii) with positive LAMP or conventional staining (CS) (Grocott methenamine silver staining or Diff-Quick™) on the basis of compatible clinical symptoms and radiologic findings. Additionally, we reviewed HIV-uninfected immunocompromised patients who underwent subcontract PCR as a historical control. LAMP was positive in 10 (90.9%) of 11 positive-CS patients. Among 13 negative-CS patients with positive LAMP, 11 (84.6%) had PCP, and the remaining 2 were categorized as having P. jirovecii colonization. LDH levels in negative-CS PCP were higher than in positive-CS PCP (p = 0.026). (1 → 3)-β-D-glucan levels in negative-CS PCP were lower than in positive-CS PCP (p = 0.011). The interval from symptom onset to diagnosis as PCP in LAMP group (3.45 ± 1.77 days; n = 22) was shorter than in subcontract PCR group (6.90 ± 2.28 days; n = 10; p < 0.001). As for patients without PCP, duration of unnecessary PCP treatment in LAMP group (2; 2-3 days; n = 10) was shorter than in subcontract PCR group (7; 7-12.25 days; n = 6; p = 0.003). LAMP showed higher sensitivity (95.4%) and positive predictive value (91.3%) than subcontract PCR did. Pneumocystis LAMP method is a sensitive and cost-effective diagnostic method and is easy to administer in general hospitals. In-house LAMP method would realize early diagnosis of PCP, resulting in improving PCP prognosis and reducing unnecessary PCP-specific treatment. PMID:25187511

  1. Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes.

    PubMed

    Mahony, James; Chong, Sylvia; Bulir, David; Ruyter, Alexandra; Mwawasi, Ken; Waltho, Daniel

    2013-08-01

    Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.

  2. Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of Dirofilaria repens from Biological Samples

    PubMed Central

    Raele, Donato Antonio; Pugliese, Nicola; Galante, Domenico; Latorre, Laura Maria; Cafiero, Maria Assunta

    2016-01-01

    Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic. PMID:27341205

  3. Development of a Sensitive Loop-Mediated Isothermal Amplification Assay That Provides Specimen-to-Result Diagnosis of Respiratory Syncytial Virus Infection in 30 Minutes

    PubMed Central

    Chong, Sylvia; Bulir, David; Ruyter, Alexandra; Mwawasi, Ken; Waltho, Daniel

    2013-01-01

    Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min. PMID:23761156

  4. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

    PubMed

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  5. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field.

  6. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    PubMed

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs. PMID:23181490

  7. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    PubMed Central

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  8. Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.

    PubMed

    Bühlmann, Andreas; Pothier, Joël F; Rezzonico, Fabio; Smits, Theo H M; Andreou, Michael; Boonham, Neil; Duffy, Brion; Frey, Jürg E

    2013-03-01

    Several molecular methods have been developed for the detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomic pipeline. The target coding sequences (CDSs) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with OptiGene Genie II chemistry and instrumentation proved to be an exceptionally rapid (under 15 min) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had an equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing.

  9. Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

    PubMed

    Bhat, A I; Siljo, A; Deeshma, K P

    2013-10-01

    The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions.

  10. Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting.

    PubMed

    Geojith, G; Dhanasekaran, S; Chandran, Salesh P; Kenneth, John

    2011-01-01

    Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay ('GenoType MTBC' from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay ('MTB LAMP') showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay ('Muniv LAMP') showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies. PMID:21047534

  11. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

  12. Integration of reverse transcriptase loop-mediated isothermal amplification with an immunochromatographic strip on a centrifugal microdevice for influenza A virus identification.

    PubMed

    Jung, J H; Park, B H; Oh, S J; Choi, G; Seo, T S

    2015-02-01

    A novel centrifugal microdevice which could perform reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and immunochromatographic strip (ICS) based amplicon detection was demonstrated for simple and cost-effective influenza A virus identification. The proposed centrifugal microdevice consists of the sample and running buffer loading reservoirs, the RT-LAMP chamber, and the ICS for detecting gene expression. The entire process could be completed sequentially and automatically by simply controlling the rotation speed and by optimizing the microfluidic design. Monoplex and multiplex RT-LAMP reactions targeting H1 and/or M gene were executed at 66 °C for 40 min, and the resultant amplicons were successfully analysed on the ICS within 15 min. Influenza A H1N1 virus was subtyped by detecting H1 and M gene on the ICS even with 10 copies of viral RNAs. Highly specific and multiplex viral typing of the integrated RT-LAMP-ICS microdevice was also demonstrated. The combination of the rapid isothermal amplification with the simple colorimetric detection on a strip in a single centrifugal microdevice will provide an advanced genetic analysis platform in the field of on-site pathogen diagnostics.

  13. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    PubMed

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

  14. Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis.

    PubMed

    Verma, Sandeep; Avishek, Kumar; Sharma, Vanila; Negi, Narendra Singh; Ramesh, Venkatesh; Salotra, Poonam

    2013-04-01

    Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7-99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6-100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9-99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1-99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL. PMID:23433714

  15. Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms

    PubMed Central

    Zhang, Chao; Yao, Yao; Zhu, Juan-Li; Zhang, Si-Nong; Zhang, Shan-Shan; Wei, Hua; Hui, Wen-Li; Cui, Ya-Li

    2016-01-01

    Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approximately 90%) in the human genome, and the demand to overcome such variation has received more attention now than ever before. The capacity to rapidly assess SNPs that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approximately 30 min under isothermal conditions. RT-LAMP amplified few copies of template to produce significant amounts of product and quantitatively detected human DNA with compatible specificity and sensitivity. The success in the establishment of this RT-LAMP protocol for CYP2C19 polymorphism testing is significant for the extension of this technique for the detection of other SNPs, which will further facilitate the development of personalized medicine. PMID:27246657

  16. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

    PubMed

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.

  17. Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  18. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence.

    PubMed

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

  19. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical.

  20. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. PMID:20403387

  1. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence

    PubMed Central

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

  2. A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool.

    PubMed

    Nzelu, Chukwunonso O; Cáceres, Abraham G; Guerrero-Quincho, Silvia; Tineo-Villafuerte, Edwin; Rodriquez-Delfin, Luis; Mimori, Tatsuyuki; Uezato, Hiroshi; Katakura, Ken; Gomez, Eduardo A; Guevara, Angel G; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2016-01-01

    Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per μl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas. PMID:26516109

  3. Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting.

    PubMed

    Geojith, G; Dhanasekaran, S; Chandran, Salesh P; Kenneth, John

    2011-01-01

    Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay ('GenoType MTBC' from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay ('MTB LAMP') showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay ('Muniv LAMP') showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.

  4. Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum.

    PubMed

    Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

    2014-01-01

    Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63 °C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

  5. A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool.

    PubMed

    Nzelu, Chukwunonso O; Cáceres, Abraham G; Guerrero-Quincho, Silvia; Tineo-Villafuerte, Edwin; Rodriquez-Delfin, Luis; Mimori, Tatsuyuki; Uezato, Hiroshi; Katakura, Ken; Gomez, Eduardo A; Guevara, Angel G; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2016-01-01

    Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per μl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas.

  6. Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish

    PubMed Central

    Saleh, Mona; Soliman, Hatem; El-Matbouli, Mansour

    2008-01-01

    Background Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel. Results A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish. Conclusion The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms. PMID:18700011

  7. Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study.

    PubMed

    Gray, Christen M; Katamba, Achilles; Narang, Pratibha; Giraldo, Jorge; Zamudio, Carlos; Joloba, Moses; Narang, Rahul; Paramasivan, C N; Hillemann, Doris; Nabeta, Pamela; Amisano, Danielle; Alland, David; Cobelens, Frank; Boehme, Catharina C

    2016-08-01

    Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary. PMID:27194691

  8. A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

    PubMed

    Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

    2016-02-01

    E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories.

  9. Multicenter evaluation of a real-time loop-mediated isothermal amplification (RealAmp) test for rapid diagnosis of Mycobacterium tuberculosis.

    PubMed

    Ou, Xichao; Wang, Shengfen; Dong, Haiyan; Pang, Yu; Li, Qiang; Xia, Hui; Qu, Yan; Zhang, Zhiying; Li, Junchen; Zhang, Jack; Huan, Shitong; Chin, Daniel P; Zhao, Yanlin

    2016-10-01

    The real-time loop-mediated isothermal amplification (RealAmp) test is a novel technology for rapid diagnosis of Mycobacterium tuberculosis (MTB) from sputum samples. This test has not previously been effectively evaluated. In this study, we assessed the performance of the RealAmp test for tuberculosis (TB) detection among TB suspected patients from three provincial hospitals in China. Each eligible TB suspected patients in Shanghai, Guangzhou, and Shandong provided one sputum specimen each, smear microscopy, liquid culture, and the RealAmp test were performed per patient. A total of 1583 TB suspected patients were enrolled in the study, and 1519 were used for the performance analysis. The sensitivity and specificity of the RealAmp test for MTB detection were calculated using liquid culture as reference method. The sensitivity of RealAmp test in smear-negative and culture-positive patients was 60.08%; the sensitivity in smear-positive and culture-positive patients was 97.09%; and the overall sensitivity in culture-positive patients was 74.88%. The specificity of RealAmp test for MTB detection was 86.50%.

  10. Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane

    PubMed Central

    Su, Yachun; Yang, Yuting; Peng, Qiong; Zhou, Dinggang; Chen, Yun; Wang, Zhuqing; Xu, Liping; Que, Youxiong

    2016-01-01

    Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg2+, primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(45) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens. PMID:27035751

  11. Multicenter evaluation of a real-time loop-mediated isothermal amplification (RealAmp) test for rapid diagnosis of Mycobacterium tuberculosis.

    PubMed

    Ou, Xichao; Wang, Shengfen; Dong, Haiyan; Pang, Yu; Li, Qiang; Xia, Hui; Qu, Yan; Zhang, Zhiying; Li, Junchen; Zhang, Jack; Huan, Shitong; Chin, Daniel P; Zhao, Yanlin

    2016-10-01

    The real-time loop-mediated isothermal amplification (RealAmp) test is a novel technology for rapid diagnosis of Mycobacterium tuberculosis (MTB) from sputum samples. This test has not previously been effectively evaluated. In this study, we assessed the performance of the RealAmp test for tuberculosis (TB) detection among TB suspected patients from three provincial hospitals in China. Each eligible TB suspected patients in Shanghai, Guangzhou, and Shandong provided one sputum specimen each, smear microscopy, liquid culture, and the RealAmp test were performed per patient. A total of 1583 TB suspected patients were enrolled in the study, and 1519 were used for the performance analysis. The sensitivity and specificity of the RealAmp test for MTB detection were calculated using liquid culture as reference method. The sensitivity of RealAmp test in smear-negative and culture-positive patients was 60.08%; the sensitivity in smear-positive and culture-positive patients was 97.09%; and the overall sensitivity in culture-positive patients was 74.88%. The specificity of RealAmp test for MTB detection was 86.50%. PMID:27425377

  12. Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification.

    PubMed

    Wu, Lan; Wang, Bo; Zhao, Mingming; Liu, Wei; Zhang, Peng; Shi, Yuhua; Xiong, Chao; Wang, Ping; Sun, Wei; Chen, Shilin

    2016-01-01

    Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong. PMID:27379153

  13. The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

    PubMed

    Wang, Yi; Wang, Yan; Ma, Aijing; Li, Dongxun; Luo, Lijuan; Liu, Dongxin; Hu, Shoukui; Jin, Dong; Liu, Kai; Ye, Changyun

    2015-12-03

    Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

  14. A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification.

    PubMed

    Aikawa, T; Horino, S; Ichihara, Y

    2015-08-01

    Severe damages to natural vegetation, agriculture, and forestry caused by overpopulation of sika deer (Cervus nippon) have markedly increased in Japan in recent years. To devise a population management plan of sika deer, information on the distribution and population size of the animal in each region is indispensable. An easy and effective method to obtain this information is to count the fecal pellets in the field. However, the habitat of sika deer in Japan overlaps that of Japanese serow (Capricornis crispus). Additionally, it is difficult to discriminate between the feces of both animals. Here, we present a rapid and precise diagnostic method for discriminating between the feces of sika deer and Japanese serow using loop-mediated isothermal amplification (LAMP) targeting cytochrome b gene in the mitochondrial DNA. Our results showed that the LAMP can discriminate between the feces of sika deer and Japanese serow, and the method is simpler and more sensitive than the conventional molecular diagnostic method. Since LAMP method does not require special skills for molecular biology techniques, even the field researchers who have never done a molecular experiment can easily carry out the protocol. In addition, the entire protocol, from DNA extraction from fecal pellet to identification of species, takes only about 75 min and does not require expensive equipment. Hence, this diagnostic method is simple, fast, and accessible to anyone. As such, the method can be a useful tool to estimate distribution and population size of sika deer.

  15. Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L

    USGS Publications Warehouse

    Shivappa, R.B.; Savan, R.; Kono, T.; Sakai, M.; Emmenegger, E.; Kurath, G.; Levine, J.F.

    2008-01-01

    Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 ??C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 10 1 TCID50 mL-1. The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV. ?? 2008 The Authors.

  16. A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

    PubMed

    Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

    2016-02-01

    E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories. PMID:26724736

  17. Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens.

    PubMed

    Gong, Jiansen; Zhuang, Linlin; Zhu, Chunhong; Shi, Shourong; Zhang, Di; Zhang, Linji; Yu, Yan; Dou, Xinhong; Xu, Bu; Wang, Chengming

    2016-04-01

    Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry. PMID:26840841

  18. Development and application of a loop-mediated isothermal amplification assay for rapid identification of aflatoxigenic molds and their detection in food samples.

    PubMed

    Luo, Jie; Vogel, Rudi F; Niessen, Ludwig

    2012-10-15

    Aflatoxins are the most thoroughly studied mycotoxins. They are produced by several members of the genus Aspergillus in section Flavi with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being frequently isolated from contaminated food sources. In this work, we describe the development and evaluation of loop-mediated isothermal amplification (LAMP) assays for rapid detection of the three species in separate analyses. The acl1-gene of A. flavus and amy1-genes of A. nomius and A. parasiticus were used as target genes. The detection limits were 2.4, 7.6 and 20pg of pure DNA/reaction for A. flavus, A. nomius and A. parasiticus, respectively. For specificity testing, DNA extracted from mycelia of representative strains of 39 Aspergillus species, 23 Penicillium species, 75 Fusarium species and 37 other fungal species was used as a template for the specific LAMP primer sets developed for the three target species. The LAMP assay was combined with a DNA extraction method for the analysis of pure fungal cultures as well as artificially contaminated Brazil nuts, peanuts and green coffee beans. It is suggested that the developed LAMP assay is a promising tool in the prediction of a potential aflatoxin risk in food and food raw materials and may therefore be suitable for high throughput analysis in the food industry. PMID:23107500

  19. Rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria by loop-mediated isothermal amplification.

    PubMed

    Cheng, Cancan; Zheng, Fen; Rui, Yongyu

    2014-12-01

    A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid detection of blaKPC, blaNDM, blaIMP, and blaVIM carbapenemase genes. Six oligonucleotides, including outer, inner, and loop primers, were designed for eight distinct regions in each target gene. Two qualitative criteria were used to evaluate LAMP reactions: visual inspection of color change and real-time detection of fluorescence change. The lower detection limit was 10 colony forming units (CFU) per reaction for real-time detection and 100 CFU per reaction for visual inspection for each gene. Two hundred twenty-two carbapenem-resistant clinical isolates (including 100 Pseudomonas aeruginosa, 100 Acinetobacter sp., and 22 Enterobacteriaceae) were tested by LAMP assay. At the same time, these isolates were confirmed by conventional polymerase chain reaction (PCR) and sequencing analysis. In these clinical isolates, the results of 11 strains with blaNDM, 11 strains with blaKPC, 11 strains with blaVIM, and 2 strains with blaIMP obtained using LAMP assays were concordant with conventional PCR. The LAMP method reported here may be a useful and powerful tool for rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria. PMID:25000338

  20. Development of a loop-mediated isothermal amplification (LAMP) for the detection of F5 fimbriae gene in enterotoxigenic Escherichia coli (ETEC).

    PubMed

    Jiang, Kuiyu; Zhu, Ying; Liu, Wenxin; Feng, Yufei; He, Lili; Guan, Weikun; Hu, Wenxia; Shi, Dongfang

    2012-11-01

    The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 10(2) cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC. PMID:22890294

  1. Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis.

    PubMed

    Draz, Mohamed Shehata; Lu, Xiaonan

    2016-01-01

    As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants. PMID:26941845

  2. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

    PubMed

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle. PMID:27150208

  3. Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane.

    PubMed

    Su, Yachun; Yang, Yuting; Peng, Qiong; Zhou, Dinggang; Chen, Yun; Wang, Zhuqing; Xu, Liping; Que, Youxiong

    2016-01-01

    Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg(2+), primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(4(5)) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens. PMID:27035751

  4. Development and Diagnostic Evaluation of Loop-Mediated Isothermal Amplification Using a New Gene Target for Rapid Detection of Helicobacter pylori

    PubMed Central

    Bakhtiari, Somaye; Alvandi, Amirhooshang; Pajavand, Hamid; Navabi, Jafar; Najafi, Farid; Abiri, Ramin

    2016-01-01

    Background Helicobacter pylori cause chronic gastritis and subsequent diseases like gastric and duodenal ulcers and gastric adenocarcinoma. Current methods for detecting H. pylori have several disadvantages and it is of utmost importance to develop a simple, quick, accurate, and cost-effective diagnostic test. Objectives The aim of this study was to set up and evaluate a diagnostic value of loop- mediated isothermal amplification (LAMP) for detecting H. pylori. Patients and Methods The analytical sensitivity values (limit of detection) of LAMP and polymerase chain reaction (PCR) were determined using serial dilutions of H. pylori DNA. Analytical specificity of the methods using new designed primers targeted ureC gene was also determined. Results The detection limits of the LAMP and PCR assay were similar and were 10 fg of pure DNA of H. pylori, which is equal to 6 copy numbers of H. pylori genome. Analytical specificity of the tests was 100% because the tests were positive only with H. pylori DNA. Conclusions The analytical sensitivity of LAMP and PCR methods, using the designed primers, was 8 times more than any other reported methods. The designed methods are specific and sensitive for detection of H. pylori in different clinical and environmental samples. PMID:27540449

  5. Loop-Mediated Isothermal Amplification Assay for Detection of Generic and Verocytotoxin-Producing Escherichia coli among Indigenous Individuals in Malaysia

    PubMed Central

    Teh, Cindy Shuan Ju; Chua, Kek Heng; Lim, Yvonne Ai Lian; Lee, Soo Ching; Thong, Kwai Lin

    2014-01-01

    We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102 CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103 CFU/mL (Tt = 31.12). We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay. PMID:24967435

  6. Rapid and effective diagnosis of pulmonary tuberculosis with novel and sensitive loop-mediated isothermal amplification (LAMP) assay in clinical samples: a meta-analysis.

    PubMed

    Yuan, Le-yong; Li, Yan; Wang, Ming; Ke, Zun-qiong; Xu, Wan-zhou

    2014-02-01

    In recent years, Loop-mediated isothermal amplification (LAMP) assay has been introduced for the diagnosis of pulmonary tuberculosis (TB). We performed the meta-analysis to establish the overall accuracy of LAMP assay for diagnosing pulmonary TB. Based on comprehensive searches of the pubmed, embase and cochrane databases, we identified outcome datas from all articles estimating diagnostic accuracy with LAMP assay until 1 October 2012. A summary estimation for sensitivity, specificity, diagnostic odds ratios (DOR) and the area under the summary ROC curve (AUC) was calculated by using the bivariate random-effects approach. The meta-analysis included 10 studies (1920 suspected specimens). The summary estimate was 80.0% (95%CI, 78.0%-83.0%) for sensitivity, 96.0% (95%CI, 95.0%-97.0%) for specificity and 119.85/0.9633 for DOR/AUC in pulmonary TB. The findings in subgroup analysis were as follows: the accuracy of LAMP assay is higher in high quality level studies than moderate and low quality level studies. The pooled sensitivity for the diagnosis of pulmonary TB was 90.0% (95%CI, 86.0-93.0%) and 75.0% (95%CI, 71.0-78.0%) for high quality level studies and moderate combined low quality level studies, respectively, while the specificity was 99.0% (95%CI, 98.0-100.0%) and 91.0% (95%CI, 88.0-94.0%). Pulmonary TB can be rapidly and accurately diagnosed with LAMP assay.

  7. Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea).

    PubMed

    Mao, Zhijuan; Qiu, Yangyu; Zheng, Lei; Chen, Jigang; Yang, Jifang

    2012-06-01

    In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.

  8. Establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of RNA from the severe fever with thrombocytopenia syndrome virus.

    PubMed

    Xu, Haihong; Zhang, Lei; Shen, Guangqiang; Feng, Cen; Wang, Xinying; Yan, Jie; Zhang, Yanjun

    2013-12-01

    As an emerging infectious disease, severe fever with thrombocytopenia syndrome virus (SFTSV) infection has been found in many areas of China. Suitable laboratory diagnostic method is urgently needed in clinical detections and epidemiological investigations. In this study, a modified, low-cost and rapid visualized one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of RNA from the SFTSV has been established. In order to avoid the risk of aerosol contamination and facilitate the naked eye to observe, a microcrystalline wax-dye capsule wrapping the highly sensitive DNA fluorescence dye SYBR Green I was added to the RT-LAMP reaction tube before the initiation of the assay. The detection limit of the established RT-LAMP assay was 10 fg template RNA per reaction mixture. The RT-LAMP assay was confirmed to be high specific to SFTSV, and no cross-reaction was found with the detection of the Chikungunya fever virus, Hemorrhagic Fever with Renal Syndrome virus (HFRSV), and Dengue fever virus. The assay was then applied for the detection of SFTSV RNA in 32 clinical serum samples and showed 94.4% consistence with the detection results of the real-time RT-PCR. The whole process, from sample preparation to result reporting, can be completed within 2h. This adapted, cost efficient and quick visualized RT-LAMP method is feasible for SFTSV field diagnosis in resource-limited field settings.

  9. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

    PubMed

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle.

  10. Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification

    PubMed Central

    Wu, Lan; Wang, Bo; Zhao, Mingming; Liu, Wei; Zhang, Peng; Shi, Yuhua; Xiong, Chao; Wang, Ping; Sun, Wei; Chen, Shilin

    2016-01-01

    Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong. PMID:27379153

  11. Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

    PubMed

    Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

    2015-06-01

    Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.

  12. Visual detection of canine parvovirus based on loop-mediated isothermal amplification combined with enzyme-linked immunosorbent assay and with lateral flow dipstick.

    PubMed

    Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

    2014-04-01

    Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP-ELISA) and with lateral flow dipstick (LAMP-LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP-ELISA and LAMP-LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP-ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP-LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR-ELISA, LAMP, LAMP-ELISA and LAMP-LFD were 10(2), 10(2), 10(-1), 10(-1) and 10(-1) TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP-ELISA and LAMP-LFD. Our data indicated that both LAMP-ELISA and LAMP-LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection.

  13. Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of Toxoplasma gondii in archived tissue samples of captive felids.

    PubMed

    Suleman, Essa; Mtshali, Moses Sibusiso; Lane, Emily

    2016-09-01

    Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and many different animals, including felids. Many molecular and serologic tests have been developed for detection of T. gondii in a wide range of hosts. Loop-mediated isothermal amplification (LAMP) is a field-friendly technique that lacks the practical drawbacks of other molecular and serologic tests, and LAMP assays have been successfully developed for detection of T. gondii in fresh tissue samples. In the current study, both a previously published and a de-novo designed LAMP assay were compared to a quantitative real-time (q)PCR assay, for the detection of T. gondii in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from captive wildlife. The LAMP assays produced conflicting results, generating both false positives and false negatives. Furthermore, the LAMP assays were unable to positively identify samples with low levels of parasites as determined by qPCR and histopathology. Therefore, these LAMP assays may not be the most suitable assays for detection of T. gondii in archived FFPE and frozen tissue samples. PMID:27449130

  14. Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences.

    PubMed

    Xu, Jinjun; Qu, Chanbao; Tao, Jianping

    2014-01-01

    Histomonas meleagridis is the causative agent of histomonosis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis, and high mortality. To develop a rapid and sensitive method for specific detection of H. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the 18S rRNA gene was established. The detection limit of the LAMP assay was 10 copies for standard plasmids containing an 18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity tests revealed that there was no cross-reaction with other protozoa such as Trichomonas gallinae, Blastocytis sp, Tetratrichomonas gallinarum, Plasmodium gallinaceum, Toxoplasma gondii, Eimeria tenella, Leucocytozoon caulleryi and Leucocytozoon sabrazesi. The assay was evaluated for its diagnostic utility using liver and caeca samples collected from suspected field cases, the detection rate was 100 and 97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification of H. meleagridis in poultry. PMID:24320623

  15. Establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of RNA from the severe fever with thrombocytopenia syndrome virus.

    PubMed

    Xu, Haihong; Zhang, Lei; Shen, Guangqiang; Feng, Cen; Wang, Xinying; Yan, Jie; Zhang, Yanjun

    2013-12-01

    As an emerging infectious disease, severe fever with thrombocytopenia syndrome virus (SFTSV) infection has been found in many areas of China. Suitable laboratory diagnostic method is urgently needed in clinical detections and epidemiological investigations. In this study, a modified, low-cost and rapid visualized one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of RNA from the SFTSV has been established. In order to avoid the risk of aerosol contamination and facilitate the naked eye to observe, a microcrystalline wax-dye capsule wrapping the highly sensitive DNA fluorescence dye SYBR Green I was added to the RT-LAMP reaction tube before the initiation of the assay. The detection limit of the established RT-LAMP assay was 10 fg template RNA per reaction mixture. The RT-LAMP assay was confirmed to be high specific to SFTSV, and no cross-reaction was found with the detection of the Chikungunya fever virus, Hemorrhagic Fever with Renal Syndrome virus (HFRSV), and Dengue fever virus. The assay was then applied for the detection of SFTSV RNA in 32 clinical serum samples and showed 94.4% consistence with the detection results of the real-time RT-PCR. The whole process, from sample preparation to result reporting, can be completed within 2h. This adapted, cost efficient and quick visualized RT-LAMP method is feasible for SFTSV field diagnosis in resource-limited field settings. PMID:23911296

  16. A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification.

    PubMed

    Aikawa, T; Horino, S; Ichihara, Y

    2015-08-01

    Severe damages to natural vegetation, agriculture, and forestry caused by overpopulation of sika deer (Cervus nippon) have markedly increased in Japan in recent years. To devise a population management plan of sika deer, information on the distribution and population size of the animal in each region is indispensable. An easy and effective method to obtain this information is to count the fecal pellets in the field. However, the habitat of sika deer in Japan overlaps that of Japanese serow (Capricornis crispus). Additionally, it is difficult to discriminate between the feces of both animals. Here, we present a rapid and precise diagnostic method for discriminating between the feces of sika deer and Japanese serow using loop-mediated isothermal amplification (LAMP) targeting cytochrome b gene in the mitochondrial DNA. Our results showed that the LAMP can discriminate between the feces of sika deer and Japanese serow, and the method is simpler and more sensitive than the conventional molecular diagnostic method. Since LAMP method does not require special skills for molecular biology techniques, even the field researchers who have never done a molecular experiment can easily carry out the protocol. In addition, the entire protocol, from DNA extraction from fecal pellet to identification of species, takes only about 75 min and does not require expensive equipment. Hence, this diagnostic method is simple, fast, and accessible to anyone. As such, the method can be a useful tool to estimate distribution and population size of sika deer. PMID:26084704

  17. Development of loop-mediated isothermal amplification and SYBR green real-time PCR methods for the detection of Citrus yellow mosaic badnavirus in citrus species.

    PubMed

    Anthony Johnson, A M; Dasgupta, I; Sai Gopal, D V R

    2014-07-01

    Citrus yellow mosaic badnavirus (CMBV) is an important pathogen in southern India spread by infected citrus propagules. One of the measures to arrest the spread of CMBV is to develop methods to screen and certify citrus propagules as CMBV-free. The methods loop-mediated isothermal amplification (LAMP) and SYBR green real-time PCR (SGRTPCR) have been developed for the efficient detection of CMBV in citrus propagules. This paper compares the sensitivities of LAMP and SGRTPCR with polymerase chain reaction (PCR) for the detection of CMBV. Whereas PCR and LAMP were able to detect CMBV from a minimum of 10 ng of total DNA of infected leaf samples, SGRTPCR could detect the same from 1 ng of total DNA. Using SGRTPCR, the viral titres were estimated to be the highest in rough lemon and lowest in Nagpur Mandarin of the five naturally infected citrus species tested. The results will help in designing suitable strategies for the sensitive detection of CMBV from citrus propagules.

  18. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil.

    PubMed

    Peng, Jun; Zhan, Yuanfeng; Zeng, Fanyun; Long, Haibo; Pei, Yuelin; Guo, Jianrong

    2013-12-01

    Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major limiting factors for watermelon production worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon in soil. Positive products were amplified only from Fon isolates and not from any other species or formae speciales of F. oxysporum tested, showing a high specificity of the primer sets. The detection limit of the RealAmp assay was 1.2 pg μL(-1) genomic DNA or 10(3) spores g(-1) of artificially inoculated soil, whereas real-time PCR could detect as low as 12 fg μL(-1) or 10(2) spores g(-1). The RealAmp assay was further applied to detect eight artificially inoculated and 85 field soil samples. No significant differences were found between the results tested by the RealAmp and real-time PCR assays. The RealAmp assay is a simple, rapid and effective technique for the quantitative detection and monitoring of Fon in soil under natural conditions.

  19. Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis

    PubMed Central

    Draz, Mohamed Shehata; Lu, Xiaonan

    2016-01-01

    As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants. PMID:26941845

  20. Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media

    PubMed Central

    Lu, Hengyu; Wilson, Bree A. L.; Ash, Gavin J.; Woruba, Sharon B.; Fletcher, Murray J.; You, Minsheng; Yang, Guang; Gurr, Geoff M.

    2016-01-01

    Phytoplasmas are insect vectored mollicutes responsible for disease in many economically important crops. Determining which insect species are vectors of a given phytoplasma is important for managing disease but is methodologically challenging because disease-free plants need to be exposed to large numbers of insects, often over many months. A relatively new method to detect likely transmission involves molecular testing for phytoplasma DNA in sucrose solution that insects have fed upon. In this study we combined this feeding medium method with a loop-mediated isothermal amplification (LAMP) assay to study 627 insect specimens of 11 Hemiptera taxa sampled from sites in Papua New Guinea affected by Bogia coconut syndrome (BCS). The LAMP assay detected phytoplasma DNA from the feeding solution and head tissue of insects from six taxa belonging to four families: Derbidae, Lophopidae, Flatidae and Ricaniidae. Two other taxa yielded positives only from the heads and the remainder tested negative. These results demonstrate the utility of combining single-insect feeding medium tests with LAMP assays to identify putative vectors that can be the subject of transmission tests and to better understand phytoplasma pathosystems. PMID:27786249

  1. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    PubMed

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

  2. Rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria by loop-mediated isothermal amplification.

    PubMed

    Cheng, Cancan; Zheng, Fen; Rui, Yongyu

    2014-12-01

    A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid detection of blaKPC, blaNDM, blaIMP, and blaVIM carbapenemase genes. Six oligonucleotides, including outer, inner, and loop primers, were designed for eight distinct regions in each target gene. Two qualitative criteria were used to evaluate LAMP reactions: visual inspection of color change and real-time detection of fluorescence change. The lower detection limit was 10 colony forming units (CFU) per reaction for real-time detection and 100 CFU per reaction for visual inspection for each gene. Two hundred twenty-two carbapenem-resistant clinical isolates (including 100 Pseudomonas aeruginosa, 100 Acinetobacter sp., and 22 Enterobacteriaceae) were tested by LAMP assay. At the same time, these isolates were confirmed by conventional polymerase chain reaction (PCR) and sequencing analysis. In these clinical isolates, the results of 11 strains with blaNDM, 11 strains with blaKPC, 11 strains with blaVIM, and 2 strains with blaIMP obtained using LAMP assays were concordant with conventional PCR. The LAMP method reported here may be a useful and powerful tool for rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria.

  3. Establishment and Application of a Loop-Mediated Isothermal Amplification Method for Simple, Specific, Sensitive and Rapid Detection of Toxoplasma gondii

    PubMed Central

    CAO, Lili; CHENG, Ronghua; YAO, Lin; YUAN, Shuxian; YAO, Xinhua

    2013-01-01

    ABSTRACT The Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with 6 primers targeting a highly conserved region of the GRA1 gene was developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondii DNA in 423 blood samples and 380 lymph node samples from 10 pig farms, and positive results were obtained for 7.8% and 8.2% of samples, respectively. The results showed that the LAMP method is slightly more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA and is expected to play an important role in clinical detection of T. gondii. PMID:23965849

  4. Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.

    PubMed

    Kanitkar, Yogendra H; Stedtfeld, Robert D; Steffan, Robert J; Hashsham, Syed A; Cupples, Alison M

    2016-01-08

    Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.

  5. Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) on a Chip from Whole Blood

    PubMed Central

    Damhorst, Gregory L.; Duarte-Guevara, Carlos; Chen, Weili; Ghonge, Tanmay; Cunningham, Brian T.; Bashir, Rashid

    2015-01-01

    Viral load measurements are an essential tool for the long-term clinical care of hum an immunodeficiency virus (HIV)-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RT-LAMP droplet, corresponding to a whole blood concentration of 670 viruses per µL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation. PMID:26705482

  6. A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

    PubMed Central

    Fernández-Soto, Pedro; Gandasegui Arahuetes, Javier; Sánchez Hernández, Alicia; López Abán, Julio; Vicente Santiago, Belén; Muro, Antonio

    2014-01-01

    Background Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries. Methodology/Principal findings A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection. Conclusions/Significance We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and

  7. Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

    PubMed Central

    Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

    2015-01-01

    Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

  8. Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

    PubMed Central

    Kanitkar, Yogendra H.; Stedtfeld, Robert D.; Steffan, Robert J.; Hashsham, Syed A.

    2016-01-01

    Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures. PMID:26746711

  9. A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips.

    PubMed

    Ahmad, Farhan; Seyrig, Gregoire; Tourlousse, Dieter M; Stedtfeld, Robert D; Tiedje, James M; Hashsham, Syed A

    2011-10-01

    Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82 dye and real time fluorescence loop-mediated isothermal amplification referred here as microRT(f)-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRT(f)-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 10(5) starting DNA copies of microRT(f)-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument. Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRT(f)-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips.

  10. Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.

    PubMed

    Kanitkar, Yogendra H; Stedtfeld, Robert D; Steffan, Robert J; Hashsham, Syed A; Cupples, Alison M

    2016-03-01

    Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures. PMID:26746711

  11. Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic.

    PubMed

    Nunes, Marcio R T; Vianez, João Lídio; Nunes, Keley N B; da Silva, Sandro Patroca; Lima, Clayton P S; Guzman, Hilda; Martins, Lívia C; Carvalho, Valéria L; Tesh, Robert B; Vasconcelos, Pedro F C

    2015-12-15

    Yellow Fever virus (YFV) is an important human pathogen in tropical areas of Africa and South America. Although an efficient vaccine is available and has been used since the early 1940s, sylvatic YFV transmission still occurs in forested areas where anthropogenic actions are present, such as mineral extraction, rearing livestock and agriculture, and ecological tourism. In this context, two distinct techniques based on the RT-PCR derived method have been previously developed, however both methods are expensive due to the use of thermo cyclers and labeled probes. We developed isothermal genome amplification, which is a rapid, sensitive, specific and low cost molecular approach for YFV genome detection. This assay used a set of degenerate primers designed for the NS1 gene and was able to amplify, within 30 min in isothermal conditions, the YFV 17D vaccine strain derived from an African wild prototype strain (Asibi), as well as field strains from Brazil, other endemic countries from South and Central America, and the Caribbean. The generic RT-LAMP assay could be helpful for YFV surveillance in field and rapid response during outbreaks in endemic areas.

  12. Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification

    PubMed Central

    2013-01-01

    Background Tick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study. Methods Primers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change. Results The sensitivity of TBEV-specific RT-LAMP assay was 102 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV. Conclusion

  13. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

    PubMed

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  14. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    PubMed Central

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  15. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

    PubMed

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  16. Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

    PubMed

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

  17. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    PubMed

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples.

  18. Loop-mediated isothermal amplification: Rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii.

    PubMed

    Mu, Xiaoqin; Nakano, Ryuichi; Nakano, Akiyo; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Tansho-Nagakawa, Shigeru; Kikuchi, Hirotoshi; Kamoshida, Go; Endo, Shiro; Yano, Hisakazu; Ono, Yasuo

    2016-02-01

    Carbapenem-resistant Acinetobacter baumannii, which are mainly induced by the production of OXA-type β-lactamases, are among the leading causes of nosocomial infections worldwide. Among the β-lactamase genes, the presence of the OXA-51-like gene carrying the upstream insertion sequence, ISAba1, was found to be one of the most prevalent carbapenem resistance mechanisms utilized by these bacteria. Consequently, it is necessary to develop a rapid detection method for ISAba1-blaOXA-51-like sequence for the timely and appropriate antibiotic treatment of A. baumannii infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was optimized for ISAba1-blaOXA-51-like detection. The LAMP primer set was designed to recognize distinct sequences in the ISAba1-blaOXA-51-like gene and could amplify the gene within 25 min at an isothermal temperature of 60°C. This LAMP assay was able to detect the ISAba1-blaOXA-51-like gene with high specificity; in addition, no cross-reactivity was observed for other types of β-lactamase producers (OXA-23-like, OXA-40-like, OXA-58-like, and IMP-1), as indicated by the absence of false positive or false negative results. The detection limit for this assay was found to be 10(0)CFU per tube which was 100-fold more sensitive than a polymerase chain reaction assay for ISAba1-blaOXA-51-like detection. Furthermore, the LAMP assay provided swift detection of the ISAba1-blaOXA-51-like gene, even directly from clinical specimens. In summary, we have described a new, rapid assay for the detection of the ISAba1-blaOXA-51-like gene from A. baumannii that could be useful in a clinical setting. This method might facilitate epidemiological studies and allow monitoring of the emergence of drug resistant strains.

  19. Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

    PubMed Central

    Dittrich, Sabine; Castonguay-Vanier, Josée; Moore, Catrin E.; Thongyoo, Narongchai; Newton, Paul N.

    2014-01-01

    Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n = 41), buffy coat specimens from R. typhi PCR-positive Lao patients (n = 42), and diverse negative controls (n = 47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n = 266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies. PMID:24371248

  20. Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

    PubMed Central

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

  1. Real-time loop-mediated isothermal amplification (LAMP) assay for group specific detection of important trichothecene producing Fusarium species in wheat.

    PubMed

    Denschlag, Carla; Rieder, Johann; Vogel, Rudi F; Niessen, Ludwig

    2014-05-01

    Trichothecene mycotoxins such as deoxynivaneol (DON), nivalenol (NIV) and T2-Toxin are produced by a variety of Fusarium spp. on cereals in the field and may be ingested by consumption of commodities and products made thereof. The toxins inhibit eukaryotic protein biosynthesis and may thus impair human and animal health. Aimed at rapid and sensitive detection of the most important trichothecene producing Fusarium spp. in a single analysis, a real-time duplex loop-mediated isothermal amplification (LAMP) assay was set up. Two sets of LAMP primers were designed independently to amplify a partial sequence of the tri6 gene in Fusarium (F.) graminearum and of the tri5 gene in Fusarium sporotrichioides, respectively. Each of the two sets detected a limited number of the established trichothecene producing Fusarium-species. However, combination of the two sets in one duplex assay enabled detection of F. graminearum, Fusarium culmorum, Fusarium cerealis, F. sporotrichioides, Fusarium langsethiae and Fusarium poae in a group specific manner. No cross reactions were detected with purified DNA from 127 other fungal species or with cereal DNA. To demonstrate the usefulness of the assay, 100 wheat samples collected from all over the German state of Bavaria were analyzed for the trichothecene mycotoxin DON by HPLC and for the presence of trichothecene producers by the new real-time duplex LAMP assay in parallel analyses. The LAMP assay showed positive results for all samples with a DON concentration exceeding 163ppb. The major advantage of the duplex LAMP assay is that the presence of six of the major trichothecene producing Fusarium spp. can be detected in a rapid and user-friendly manner with only one single assay. To our knowledge this is the first report of the use of a multiplex LAMP assay for fungal organisms.

  2. Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification.

    PubMed

    Komatsu, Ken; Urayama, Syun-Ichi; Katoh, Yu; Fuji, Shin-Ichi; Hase, Shu; Fukuhara, Toshiyuki; Arie, Tsutomu; Teraoka, Tohru; Moriyama, Hiromitsu

    2016-02-01

    Magnaporthe oryzae chrysovirus 1 (MoCV1) is a mycovirus with a dsRNA genome that infects the rice blast fungus Magnaporthe oryzae and impairs its growth. To date, MoCV1 has only been found in Vietnamese isolates of M. oryzae, and the distribution of this virus in M. oryzae isolates from other parts of the world remains unknown. In this study, using a one-step reverse transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus in M. oryzae in Japan (named MoCV1-AK) whose sequence shares considerable similarity with that of the MoCV1 Vietnamese isolate. To establish a system for a comprehensive survey of MoCV1 infection in the field, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. The sensitivity of the RT-LAMP assay was at least as high as that of the one-step RT-PCR assay. In addition, we detected MoCV1-AK in M. oryzae-infected oatmeal agar plates and lesions on rice leaves using the RT-LAMP assay without dsRNA extraction, by simple sampling with a toothpick. Preliminary screening of MoCV1 in Japanese M. oryzae isolates indicated that MoCV1 is currently distributed in rice fields in Japan. Our results provide a first example of the application of RT-LAMP for the detection of mycoviruses, which will accelerate surveys for mycovirus infection. PMID:26547578

  3. Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: loop-mediated isothermal amplification for malaria diagnosis.

    PubMed

    Ghayour Najafabadi, Zahra; Oormazdi, Hormozd; Akhlaghi, Lame; Meamar, Ahmad Reza; Nateghpour, Mehdi; Farivar, Leila; Razmjou, Elham

    2014-08-01

    This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к=0.64) and poor to fair for saliva LAMP and urine LAMP (к=0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.

  4. Evaluation of three enzyme immunoassays and a loop-mediated isothermal amplification test for the laboratory diagnosis of Clostridium difficile infection.

    PubMed

    Bruins, M J; Verbeek, E; Wallinga, J A; Bruijnesteijn van Coppenraet, L E S; Kuijper, E J; Bloembergen, P

    2012-11-01

    The laboratory diagnosis of Clostridium difficile infection (CDI) consists of the detection of toxigenic Clostridium difficile, and/or its toxins A or B in stool preferably in a two-step algorithm. In a prospective study, we compared the performance of three toxin enzyme immunoassays (EIAs)-ImmunoCard Toxins A & B, Premier Toxins A & B and C. diff Quik Chek Complete, which combines a toxins test and a glutamate dehydrogenase (GDH) antigen EIA in one device -and the loop-mediated isothermal amplification assay Illumigene C. difficile. In total 986 stool samples were analyzed. Compared with toxigenic culture as the gold standard, sensitivities, specificities, PPV and NPV values of the toxin EIAs were 41.1-54.8 %, 98.9-100 %, 75.0-100 % and 95.5-96.5 % respectively, of the Illumigene assay 93.3 %, 99.7 %, 95.8 % and 99.5 %. Illumigene assays performed significantly better for non-014/020 PCR-ribotypes than for C. difficile isolates belonging to 014/020. Discrepant analysis of three culture-negative, but Illumigene-positive samples, revealed the presence of toxin genes using real-time PCRs. In addition to the GDH EIA (NPV of 99.8 %), the performance of Illumigene allows this test to be introduced as a first screening test for CDI- or as a confirmation test for GDH -positive samples, although the initial invalid Illumigene result of 4.4 % is a point of concern.

  5. A nationwide survey of pathogenic leptospires in urine of cattle and buffaloes by Loop-mediated isothermal amplification (LAMP) method in Thailand, 2011–2013

    PubMed Central

    SUWANCHAROEN, Duangjai; LIMLERTVATEE, Supaluck; CHETIYAWAN, Philaiphon; TONGPAN, Phichet; SANGKAEW, Nongluck; SAWADDEE, Yaowarat; INTHAKAN, Kanya; WIRATSUDAKUL, Anuwat

    2016-01-01

    Leptospirosis is a worldwide distributed zoonosis which has long been endemic in Thailand. Cattle and buffaloes are important livestock species that live in close contact with humans, especially in rural areas. These animals may, therefore, act as long-term carriers of leptospirosis for humans and other livestock species. The present study employed loop-mediated isothermal amplification (LAMP) method to detect pathogenic leptospiral 16S rDNA in the urine of cattle and buffaloes for assessing associations between uroprevalence and species, sex, age and spatial distribution. A total of 3,657 urine samples were collected for laboratory diagnosis, and 312 of which turned positive to the test (true prevalence 5.90%; 95% CI 4.98–6.91). The highest true uroprevalence was found in lower northern region at 19.80% (95% CI 15.83–24.32) followed by upper and lower northeastern regions at 15.22% and 6.25%, respectively. However, the highest true uroprevalence in beef cattle, the majority of cattle in Thailand, was recorded in northeastern region which is the endemic area of human leptospirosis. The uroprevalence was not statistically different among species and types of examined animals. Male animals were over twice more likely to be infected compared to females. Excluding animals younger than one year of age due to small sample size, the uroprevalence upraised with increasing age. A collaborative investigation between veterinary and public health sectors is required to holistically explore the link between leptospirosis in humans and livestock, especially in high prevalent areas. PMID:27302016

  6. Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

    PubMed

    Pinhanelli, V C; Costa, P N M; Silva, G; Aguiar, D M; Silva, C M L; Fachin, A L; Marins, M

    2015-01-01

    Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME. PMID:26782434

  7. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    PubMed

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples. PMID:27376196

  8. Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

    PubMed

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

  9. Evaluation of real-time loop-mediated isothermal amplification (RealAmp) for rapid detection of Mycobacterium tuberculosis from sputum samples.

    PubMed

    Li, Yiming; Shi, Lei; Pan, Anqi; Cao, Weiwei; Chen, Xun; Meng, Hecheng; Yan, He; Miyoshi, Shin-ichi; Ye, Lei

    2014-09-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) leads to serious health problems as a chronic respiratory infectious disease. Here we established a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) using a portable ESE Quant tube scanner as a convenient rapid detection method for MTB. The method efficacy from sputum samples was further investigated, and the reaction time was only 20min with the detection limit low to 10(2)CFU/ml concentration of MTB. We assessed a total of 1067 samples by the RealAmp assay, comparing the results with smear microscopy and conventional culture methods. To examine whether the failure to detect TB by culturing is due to low sensitivity or true absence, we examined the culture negative samples by commercial real time PCR MTB detection kit, and the results were compared with RealAmp. The data showed that RealAmp assay had a higher positive rate than that of sputum smear and culture methods. RealAmp had a sensitivity of 96.70% and a specificity of 91.55% when compared with culture. In addition, its sensitivity and specificity were 95.29% and 86.88% respectively compared with examination of smear samples using light microscopy. The sensitivity of RealAmp in comparison to real time PCR was 98.25% and specificity was 99.11% in validation of culture negative samples. The present study revealed the newly established RealAmp assay as a convenient, efficient, sensitive and specific method that could be an alternative for rapid detection of MTB and a tool to validate culture and smear negative samples. Furthermore, the portability of the ESE Quant tube scanner also contributed to the promising application for grassroots and field detection of MTB.

  10. Loop-mediated isothermal amplification for Rickettsia typhi (the causal agent of murine typhus): problems with diagnosis at the limit of detection.

    PubMed

    Dittrich, Sabine; Castonguay-Vanier, Josée; Moore, Catrin E; Thongyoo, Narongchai; Newton, Paul N; Paris, Daniel H

    2014-03-01

    Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n=41), buffy coat specimens from R. typhi PCR-positive Lao patients (n=42), and diverse negative controls (n=47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n=266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies.

  11. Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

    PubMed

    Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

    2016-08-01

    Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. PMID:27054888

  12. Rapid and Sensitive Detection of Bartonella bacilliformis in Experimentally Infected Sand Flies by Loop-Mediated Isothermal Amplification (LAMP) of the Pap31 Gene

    PubMed Central

    Angkasekwinai, Nasikarn; Atkins, Erin H.; Johnson, Richard N.; Grieco, John P.; Ching, Wei Mei; Chao, Chien Chung

    2014-01-01

    Background Carrion' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis. Methods and Findings The sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis. Conclusions The LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector. PMID:25522230

  13. A new surveillance and response tool: risk map of infected Oncomelania hupensis detected by Loop-mediated isothermal amplification (LAMP) from pooled samples.

    PubMed

    Tong, Qun-Bo; Chen, Rui; Zhang, Yi; Yang, Guo-Jing; Kumagai, Takashi; Furushima-Shimogawara, Rieko; Lou, Di; Yang, Kun; Wen, Li-Yong; Lu, Shao-Hong; Ohta, Nobuo; Zhou, Xiao-Nong

    2015-01-01

    Although schistosomiasis remains a serious health problem worldwide, significant achievements in schistosomiasis control has been made in the People's Republic of China. The disease has been eliminated in five out of 12 endemic provinces, and the prevalence in remaining endemic areas is very low and is heading toward elimination. A rapid and sensitive method for monitoring the distribution of infected Oncomelania hupensis is urgently required. We applied a loop-mediated isothermal amplification (LAMP) assay targeting 28S rDNA for the rapid and effective detection of Schistosoma japonicum DNA in infected and prepatent infected O. hupensis snails. The detection limit of the LAMP method was 100 fg of S. japonicum genomic DNA. To promote the application of the approach in the field, the LAMP assay was used to detect infection in pooled samples of field-collected snails. In the pooled sample detection, snails were collected from 28 endemic areas, and 50 snails from each area were pooled based on the maximum pool size estimation, crushed together and DNA was extracted from each pooled sample as template for the LAMP assay. Based on the formula for detection from pooled samples, the proportion of positive pooled samples and the positive proportion of O. hupensis detected by LAMP of Xima village reached 66.67% and 1.33%, while those of Heini, Hongjia, Yangjiang and Huangshan villages were 33.33% and 0.67%, and those of Tuanzhou and Suliao villages were 16.67% and 0.33%, respectively. The remaining 21 monitoring field sites gave negative results. A risk map for the transmission of schistosomiasis was constructed using ArcMap, based on the positive proportion of O. hupensis infected with S. japonicum, as detected by the LAMP assay, which will form a guide for surveillance and response strategies in high risk areas.

  14. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-01

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

  15. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-01

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA. PMID:26980448

  16. The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification (LAMP) Assay for Tuberculosis Diagnosis in Adults with Chronic Cough in Malawi

    PubMed Central

    Nliwasa, Marriott; MacPherson, Peter; Chisala, Palesa; Kamdolozi, Mercy; Khundi, McEwen; Kaswaswa, Kruger; Mwapasa, Mphatso; Msefula, Chisomo; Sohn, Hojoon; Flach, Clare; Corbett, Elizabeth L.

    2016-01-01

    Background Current tuberculosis diagnostics lack sensitivity, and are expensive. Highly accurate, rapid and cheaper diagnostic tests are required for point of care use in low resource settings with high HIV prevalence. Objective To investigate the sensitivity and specificity, and cost of loop-mediated isothermal amplification (LAMP) assay for tuberculosis diagnosis in adults with chronic cough compared to Xpert® MTB/RIF, fluorescence smear microscopy. Methods Between October 2013 and March 2014, consecutive adults at a primary care clinic were screened for cough, offered HIV testing and assessed for tuberculosis using LAMP, Xpert® MTB/RIF and fluorescence smear microscopy. Sensitivity and specificity (with culture as reference standard), and costs were estimated. Results Of 273 adults recruited, 44.3% (121/273) were HIV-positive and 19.4% (53/273) had bacteriogically confirmed tuberculosis. The sensitivity of LAMP compared to culture was 65.0% (95% CI: 48.3% to 79.4%) with 100% (95% CI: 98.0% to 100%) specificity. The sensitivity of Xpert® MTB/RIF (77.5%, 95% CI: 61.5% to 89.2%) was similar to that of LAMP, p = 0.132. The sensitivity of concentrated fluorescence smear microscopy with routine double reading (87.5%, 95% CI: 73.2% to 95.8%) was higher than that of LAMP, p = 0.020. All three tests had high specificity. The lowest cost per test of LAMP was at batch size of 14 samples (US$ 9.98); this was lower than Xpert® MTB/RIF (US$ 13.38) but higher than fluorescence smear microscopy (US$ 0.65). Conclusion The sensitivity of LAMP was similar to Xpert® MTB/RIF but lower than fluorescence smear microscopy; all three tests had high specificity. These findings support the Malawi policy that recommends a combination of fluorescence smear microscopy and Xpert® MTB/RIF prioritised for people living with HIV, already found to be smear-negative, or being considered for retreatment of tuberculosis. PMID:27171380

  17. Simple and rapid method for the detection of Filobasidiella neoformans in a probiotic dairy product by using loop-mediated isothermal amplification.

    PubMed

    Ishikawa, Hiroshi; Kasahara, Kohei; Sato, Sumie; Shimakawa, Yasuhisa; Watanabe, Koichi

    2014-05-16

    Yeast contamination is a serious problem in the food industry and a major cause of food spoilage. Several yeasts, such as Filobasidiella neoformans, which cause cryptococcosis in humans, are also opportunistic pathogens, so a simple and rapid method for monitoring yeast contamination in food is essential. Here, we developed a simple and rapid method that utilizes loop-mediated isothermal amplification (LAMP) for the detection of F. neoformans. A set of five specific LAMP primers was designed that targeted the 5.8S-26S rDNA internal transcribed spacer 2 region of F. neoformans, and the primer set's specificity was confirmed. In a pure culture of F. neoformans, the LAMP assay had a lower sensitivity threshold of 10(2)cells/mL at a runtime of 60min. In a probiotic dairy product artificially contaminated with F. neoformans, the LAMP assay also had a lower sensitivity threshold of 10(2)cells/mL, which was comparable to the sensitivity of a quantitative PCR (qPCR) assay. We also developed a simple two-step method for the extraction of DNA from a probiotic dairy product that can be performed within 15min. This method involves initial protease treatment of the test sample at 45°C for 3min followed by boiling at 100°C for 5min under alkaline conditions. In a probiotic dairy product artificially contaminated with F. neoformans, analysis by means of our novel DNA extraction method followed by LAMP with our specific primer set had a lower sensitivity threshold of 10(3)cells/mL at a runtime of 60min. In contrast, use of our novel method of DNA extraction followed by qPCR assay had a lower sensitivity threshold of only 10(5)cells/mL at a runtime of 3 to 4h. Therefore, unlike the PCR assay, our LAMP assay can be used to quickly evaluate yeast contamination and is sensitive even for crude samples containing bacteria or background impurities. Our study provides a powerful tool for the primary screening of large numbers of food samples for yeast contamination.

  18. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40 - 50 million annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal a...

  19. Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification.

    PubMed

    Pillai, D; Bonami, J-R; Sri Widada, J

    2006-05-01

    A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product.

  20. [Detection of JAK2V617F mutation rate by real-time fluorescent quantitative PCR using allele specific primer and TaqMan-MGB probe for dual inhibiting amplification of wild type alleles].

    PubMed

    Liang, Guo-Wei; Shao, Dong-Hua; He, Mei-Ling; Cao, Qing-Yun

    2012-12-01

    This study was purposed to develop a real-time PCR assay for sensitive quantification of JAK2V617F allele burden in peripheral blood and to evaluate the clinical value of this method. Both allele-specific mutant reverse primer and wild-type TaqMan-MGB probe were used for dual-inhibiting amplification of wild-type alleles in a real-time PCR, and then the JAK2V617F mutant alleles were amplified specially. The standard curve for quantification of JAK2V617F was established by percentages of JAK2V617F alleles with threshold cycle (Ct) values in a real-time PCR. Furthermore, 89 apparent healthy donors were tested by this method. The results showed that the quantitative lower limit of this method for JAK2V617F was 0.1%, and the intra- and inter-assay average variability for quantifying percentage of JAK2V617F in total DNA was 4.1% and 6.1%, respectively. Two JAK2V617F-positive individuals were identified (the percentage of JAK2V617F alleles were 0.64% and 0.98%, respectively) using this method in blood from 89 apparently healthy donors. It is concluded that the developed method with highly sensitive and reproducible quantification of JAK2V617F mutant burden can be used clinically for diagnosis and evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms. Moreover, this technique can be also used for quantitative detection of variety of single nucleotide mutation.

  1. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia.

    PubMed

    Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad; Pournia, Yadollah; Zebardast, Nozhat; Kazemi, Bahram

    2014-07-01

    Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM-, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia.

  2. Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea.

    PubMed

    Kurosaki, Yohei; Magassouba, N'Faly; Oloniniyi, Olamide K; Cherif, Mahamoud S; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

    2016-02-01

    Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure.

  3. Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

    PubMed Central

    Kurosaki, Yohei; Magassouba, N’Faly; Oloniniyi, Olamide K.; Cherif, Mahamoud S.; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

    2016-01-01

    Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure. PMID:26900929

  4. Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil

    PubMed Central

    Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China. PMID:24376590

  5. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

    PubMed

    Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

  6. Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection

    PubMed Central

    Waters, Ryan A.; Fowler, Veronica L.; Armson, Bryony; Nelson, Noel; Gloster, John; Paton, David J.; King, Donald P.

    2014-01-01

    Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health. PMID:25165973

  7. SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification.

    PubMed

    Nawattanapaiboon, Kawin; Kiatpathomchai, Wansika; Santanirand, Pitak; Vongsakulyanon, Apirom; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Srikhirin, Toemsak

    2015-12-15

    In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA.

  8. Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1.

    PubMed

    Rathinasabapathi, P; Hiremath, Deepak S; Arunraj, Rex; Parani, M

    2015-12-01

    New Delhi metallo-β-lactamase-1 gene (bla NDM-1 ) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of β-lactam ring, and provides resistance against major classes of β-lactam antibiotics. Dissemination of the plasmid borne bla NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla NDM-1 . Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6-25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla NDM-1 gene.

  9. Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

    PubMed

    Lim, Hazel Sin Yue; Zheng, Qianwang; Miks-Krajnik, Marta; Turner, Matthew; Yuk, Hyun-Gyun

    2015-06-01

    The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in

  10. Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.

    PubMed

    Zhou, Qian-Jin; Wang, Lei; Chen, Jiong; Wang, Rui-Na; Shi, Yu-Hong; Li, Chang-Hong; Zhang, De-Min; Yan, Xiao-Jun; Zhang, Yan-Jun

    2014-09-01

    Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.

  11. Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts.

    PubMed

    Luo, Jie; Taniwaki, Marta H; Iamanaka, Beatriz T; Vogel, Rudi F; Niessen, Ludwig

    2014-02-17

    Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 10⁵ conidia per reaction with the primer set ID9 for A. nomius and 10⁴ conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 10¹ and 10² conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61

  12. Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts.

    PubMed

    Luo, Jie; Taniwaki, Marta H; Iamanaka, Beatriz T; Vogel, Rudi F; Niessen, Ludwig

    2014-02-17

    Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 10⁵ conidia per reaction with the primer set ID9 for A. nomius and 10⁴ conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 10¹ and 10² conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61

  13. SNP-Based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing®.

    PubMed

    Busato, Florence; Tost, Jörg

    2015-01-01

    The analysis of allele-specific DNA methylation patterns has recently attracted much interest as loci of allele-specific DNA methylation overlap with known risk loci for complex diseases and the analysis might contribute to the fine-mapping and interpretation of non-coding genetic variants associated with complex diseases and improve the understanding between genotype and phenotype. In the presented protocol, we present a method for the analysis of DNA methylation patterns on both alleles separately using heterozygous Single Nucleotide Polymorphisms (SNPs) as anchor for allele-specific PCR amplification followed by analysis of the allele-specific DNA methylation patterns by Pyrosequencing(®). Pyrosequencing is an easy-to-handle, quantitative real-time sequencing method that is frequently used for genotyping as well as for the analysis of DNA methylation patterns. The protocol consists of three major steps: (1) identification of individuals heterozygous for a SNP in a region of interest using Pyrosequencing; (2) analysis of the DNA methylation patterns surrounding the SNP on bisulfite-treated DNA to identify regions of potential allele-specific DNA methylation; and (3) the analysis of the DNA methylation patterns associated with each of the two alleles, which are individually amplified using allele-specific PCR. The enrichment of the targeted allele is re-enforced by modification of the allele-specific primers at the allele-discriminating base with Locked Nucleic Acids (LNA). For the proof-of-principle of the developed approach, we provide assay details for three imprinted genes (IGF2, IGF2R, and PEG3) within this chapter. The mean of the DNA methylation patterns derived from the individual alleles corresponds well to the overall DNA methylation patterns and the developed approach proved more reliable compared to other protocols for allele-specific DNA methylation analysis.

  14. Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches.

    PubMed

    Zhang, Shu-Qin; Tan, Bin; Li, Peng; Wang, Feng-Xue; Guo, Li; Yang, Yong; Sun, Na; Zhu, Hong-Wei; Wen, Yong-Jun; Cheng, Shi-Peng

    2014-10-01

    Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 10(1) copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials.

  15. Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

    PubMed Central

    Orue, Andrea; Rieber, Manuel

    2016-01-01

    Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting. PMID:27632281

  16. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    PubMed

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR. PMID:12470637

  17. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    PubMed

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

  18. Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.

    PubMed

    Mokrousov, Igor; Otten, Tatiana; Vyshnevskiy, Boris; Narvskaya, Olga

    2003-07-01

    We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB. PMID:12821473

  19. Enhancing allele-specific PCR for specifically detecting short deletion and insertion DNA mutations.

    PubMed

    Wang, Yiran; Rollin, Joseph A; Zhang, Y-H Percival

    2010-02-01

    Allele-specific PCR (AS-PCR) has been widely used for the detection of single nucleotide polymorphism. But there are some challenges in using AS-PCR for specifically detecting DNA variations with short deletions or insertions. The challenges are associated with designing selective allele-specific primers as well as the specificity of AS-PCR in distinguishing some types of single base-pair mismatches. In order to address such problems and enhance the applicability of AS-PCR, a general primer design method was developed to create a multiple base-pair mismatch between the primer 3'-terminus and the template DNA. This approach can destabilize the primer-template complex more efficiently than does a single base-pair mismatch, and can dramatically increase the specificity of AS-PCR. As a proof-of-principle demonstration, the method of primer design was applied in colony PCR for identifying plasmid DNA deletion or insertion mutants after site-directed mutagenesis. As anticipated, multiple base-pair mismatches achieved much more specific PCR amplification than single base-pair mismatches. Therefore, with the proposed primer design method, the detection of short nucleotide deletion and insertion mutations becomes simple, accurate and more reliable.

  20. Allele-specific MMP-3 transcription under in vivo conditions

    SciTech Connect

    Zhu Chaoyong; Odeberg, Jacob; Hamsten, Anders; Eriksson, Per . E-mail: Per.Eriksson@ki.se

    2006-09-29

    A common matrix metalloproteinases-3 (MMP-3) -1612 5A/6A promoter polymorphism is associated with risk for cardiovascular disease, rheumatoid arthritis, and other diseases. Here we used the haplotype chromatin immunoprecipitation method to study allele-specific MMP-3 expression under in vivo conditions in heterozygous THP-1 cells. Pyrosequencing was used to analyse the ratio of 5A-allele to 6A-allele after chromatin immunoprecipitation using an antibody against phosphorylated active RNA polymerase II. There was no allele-specific difference in transcriptional activity during basal conditions, i.e., in unstimulated monocytic THP-1 cells. However, after stimulation of MMP-3 expression by monocyte differentiation or incubation with IL-1{beta}, the haplotype containing the 5A-allele was associated with higher transcriptional activity compared with the 6A-containing haplotype. Electromobility shift assay demonstrated increased binding of nuclear proteins to the 5A-allele after monocyte differentiation. In conclusion, the common MMP-3 5A/6A promoter polymorphism appears to be functional only during specific environmental conditions involving inflammation.

  1. Allele-Specific DNA Methylation Detection by Pyrosequencing®.

    PubMed

    Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide information on the methylation status of individual alleles of genes. This information may be of importance in many situations. In particular, in cancer both alleles of tumour suppressor genes generally need to be inactivated for a phenotypic effect to be observed. Here, we present a simple and cost-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon. PMID:26103906

  2. Delimiting Allelic Imbalance of TYMS by Allele-Specific Analysis

    PubMed Central

    Balboa-Beltrán, Emilia; Cruz, Raquel; Carracedo, Angel; Barros, Francisco

    2015-01-01

    Abstract Allelic imbalance of thymidylate synthase (TYMS) is attributed to polymorphisms in the 5′- and 3′-untranslated region (UTR). These polymorphisms have been related to the risk of suffering different cancers, for example leukemia, breast or gastric cancer, and response to different drugs, among which are methotrexate glutamates, stavudine, and specifically 5-fluorouracil (5-FU), as TYMS is its direct target. A vast literature has been published in relation to 5-FU, even suggesting the sole use of these polymorphisms to effectively manage 5-FU dosage. Estimates of the extent to which these polymorphisms influence in TYMS expression have in the past been based on functional analysis by luciferase assays and quantification of TYMS mRNA, but both these studies, as the association studies with cancer risk or with toxicity or response to 5-FU, are very contradictory. Regarding functional assays, the artificial genetic environment created in luciferase assay and the problems derived from quantitative polymerase chain reactions (qPCRs), for example the use of a reference gene, may have distorted the results. To avoid these sources of interference, we have analyzed the allelic imbalance of TYMS by allelic-specific analysis in peripheral blood mononuclear cells (PBMCs) from patients. Allelic imbalance in PBMCs, taken from 40 patients with suspected myeloproliferative haematological diseases, was determined by fluorescent fragment analysis (for the 3′-UTR polymorphism), Sanger sequencing and allelic-specific qPCR in multiplex (for the 5′-UTR polymorphisms). For neither the 3′- nor the 5′-UTR polymorphisms did the observed allelic imbalance exceed 1.5 fold. None of the TYMS polymorphisms is statistically associated with allelic imbalance. The results acquired allow us to deny the previously established assertion of an influence of 2 to 4 fold of the rs45445694 and rs2853542 polymorphisms in the expression of TYMS and narrow its allelic imbalance to 1.5 fold

  3. Genomic landscape of human allele-specific DNA methylation.

    PubMed

    Fang, Fang; Hodges, Emily; Molaro, Antoine; Dean, Matthew; Hannon, Gregory J; Smith, Andrew D

    2012-05-01

    DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for full-genome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species. PMID:22523239

  4. Allele-specific chemical genetics: concept, strategies, and applications.

    PubMed

    Islam, Kabirul

    2015-02-20

    The relationship between DNA and protein sequences is well understood, yet because the members of a protein family/subfamily often carry out the same biochemical reaction, elucidating their individual role in cellular processes presents a challenge. Forward and reverse genetics have traditionally been employed to understand protein functions with considerable success. A fundamentally different approach that has gained widespread application is the use of small organic molecules, known as chemical genetics. However, the slow time-scale of genetics and inherent lack of specificity of small molecules used in chemical genetics have limited the applicability of these methods in deconvoluting the role of individual proteins involved in fast, dynamic biological events. Combining the advantages of both the techniques, the specificity achieved with genetics along with the reversibility and tunability of chemical genetics, has led to the development of a powerful approach to uncover protein functions in complex biological processes. This technique is known as allele-specific chemical genetics and is rapidly becoming an essential toolkit to shed light on proteins and their mechanism of action. The current review attempts to provide a comprehensive description of this approach by discussing the underlying principles, strategies, and successful case studies. Potential future implications of this technology in expanding the frontiers of modern biology are discussed.

  5. Extensive allele-specific translational regulation in hybrid mice.

    PubMed

    Hou, Jingyi; Wang, Xi; McShane, Erik; Zauber, Henrik; Sun, Wei; Selbach, Matthias; Chen, Wei

    2015-08-07

    Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression.

  6. Genomic landscape of human allele-specific DNA methylation

    PubMed Central

    Fang, Fang; Hodges, Emily; Molaro, Antoine; Dean, Matthew; Hannon, Gregory J.; Smith, Andrew D.

    2012-01-01

    DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for full-genome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species. PMID:22523239

  7. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing.

    PubMed

    Shen, Ronglai; Seshan, Venkatraman E

    2016-09-19

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of 'actionable' mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel.

  8. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing

    PubMed Central

    Shen, Ronglai; Seshan, Venkatraman E.

    2016-01-01

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of ‘actionable’ mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel. PMID:27270079

  9. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing.

    PubMed

    Shen, Ronglai; Seshan, Venkatraman E

    2016-09-19

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of 'actionable' mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel. PMID:27270079

  10. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  11. Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris.

    PubMed

    Amarasinghe, Harindra E; Toghill, Bradley J; Nathanael, Despina; Mallon, Eamonn B

    2015-01-01

    Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes.

  12. DNA bending potentials for loop-mediated nucleosome repositioning

    NASA Astrophysics Data System (ADS)

    Biswas, M.; Wocjan, T.; Langowski, J.; Smith, J. C.

    2012-02-01

    Nucleosome repositioning is a fundamental process in gene function. DNA elasticity is a key element of loop-mediated nucleosome repositioning. Two analytical models for DNA elasticity have been proposed: the linear sub-elastic chain (SEC), which allows DNA kinking, and the worm-like chain (WLC), with a harmonic bending potential. In vitro studies have shown that nucleosomes reposition in a discontiguous manner on a segment of DNA and this has also been found in ground-state calculations with the WLC analytical model. Here we study using Monte Carlo simulation the dynamics of DNA loop-mediated nucleosome repositioning at physiological temperatures using the SEC and WLC potentials. At thermal energies both models predict nearest-neighbor repositioning of nucleosomes on DNA, in contrast to the repositioning in jumps observed in experiments. This suggests a crucial role of DNA sequence in nucleosome repositioning.

  13. DNA bending potentials for loop-mediated nucleosome repositioning

    SciTech Connect

    Langowski, Jorg

    2012-01-01

    Nucleosome repositioning is a fundamental process in gene function. DNA elasticity is a key element of loop-mediated nucleosome repositioning. Two analytical models for DNA elasticity have been proposed: the linear sub-elastic chain (SEC), which allows DNA kinking, and the worm-like chain (WLC), with a harmonic bending potential. In vitro studies have shown that nucleosomes reposition in a discontiguous manner on a segment of DNA and this has also been found in ground-state calculations with the WLC analytical model. Here we study using Monte Carlo simulation the dynamics of DNA loop-mediated nucleosome repositioning at physiological temperatures using the SEC and WLC potentials. At thermal energies both models predict nearest-neighbor repositioning of nucleosomes on DNA, in contrast to the repositioning in jumps observed in experiments. This suggests a crucial role of DNA sequence in nucleosome repositioning.

  14. Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

    PubMed

    Liu, Ai Hong; Guan, Gui Quan; Liu, Jun Long; Liu, Zhi Jie; Leblanc, Neil; Li, You Quan; Gao, Jin Liang; Ma, Mi Ling; Niu, Qing Li; Ren, Qiao Yun; Bai, Qi; Yin, Hong; Luo, Jian Xun

    2011-02-01

    Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

  15. Self-(in)compatibility inheritance and allele-specific marker development in yellow mustard (Sinapis alba).

    PubMed

    Zeng, Fangqin; Cheng, Bifang

    2014-01-01

    Yellow mustard (Sinapis alba) has a sporophytic self-incompatibility reproduction system. Genetically stable self-incompatible (SI) and self-compatible (SC) inbred lines have recently been developed in this crop. Understanding the S haplotype of different inbred lines and the inheritance of the self-(in)compatibility (SI/SC) trait is very important for breeding purposes. In this study, we used the S-locus gene-specific primers in Brassica rapa and Brassica oleracea to clone yellow mustard S-locus genes of SI lines Y514 and Y1130 and SC lines Y1499 and Y1501. The PCR amplification results and DNA sequences of the S-locus genes revealed that Y514 carried the class I S haplotype, while Y1130, Y1499, and Y1501 had the class II S haplotype. The results of our genetic studies indicated that self-incompatibility was dominant over self-compatibility and controlled by a one-gene locus in the two crosses of Y514 × Y1499 and Y1130 × Y1501. Of the five S-locus gene polymorphic primer pairs, Sal-SLGI and Sal-SRKI each generated one dominant marker for the SI phenotype of Y514; Sal-SLGII and Sal-SRKII produced dominant marker(s) for the SC phenotype of Y1501 and Y1499; Sal-SP11II generated one dominant marker for Y1130. These markers co-segregated with the SI/SC phenotype in the F2 populations of the two crosses. In addition, co-dominant markers were developed by mixing the two polymorphic primer pairs specific for each parent in the multiplex PCR, which allowed zygosity to be determined in the F2 populations. The SI/SC allele-specific markers have proven to be very useful for the selection of the desirable SC genotypes in our yellow mustard breeding program.

  16. A computational workflow to identify allele-specific expression and epigenetic modification in maize.

    PubMed

    Wei, Xiaoxing; Wang, Xiangfeng

    2013-08-01

    Allele-specific expression refers to the preferential expression of one of the two alleles in a diploid genome, which has been thought largely attributable to the associated cis-element variation and allele-specific epigenetic modification patterns. Allele-specific expression may contribute to the heterosis (or hybrid vigor) effect in hybrid plants that are produced from crosses of closely-related species, subspecies and/or inbred lines. In this study, using Illumina high-throughput sequencing of maize transcriptomics, chromatic H3K27me3 histone modification and DNA methylation data, we developed a new computational framework to identify allele-specifically expressed genes by simultaneously tracking allele-specific gene expression patterns and the epigenetic modification landscape in the seedling tissues of hybrid maize. This approach relies on detecting nucleotide polymorphisms and any genomic structural variation between two parental genomes in order to distinguish paternally or maternally derived sequencing reads. This computational pipeline also incorporates a modified Chi-square test to statistically identify allele-specific gene expression and epigenetic modification based on the Poisson distribution.

  17. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing.

    PubMed

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J; Szatkiewicz, Jin P

    2015-08-18

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

  18. A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference.

    PubMed

    Takahashi, Masaki; Hohjoh, Hirohiko

    2014-11-01

    Allele-specific silencing by RNA interference (ASP-RNAi) is an atypical RNAi that is capable of discriminating target alleles from non-target alleles, and may be therapeutically useful for specific inhibition of disease-causing alleles without affecting their corresponding normal alleles. However, it is difficult to design and select small interfering RNA (siRNAs) that confer ASP-RNAi. A major problem is that there are few appropriate measures in determining optimal allele-specific siRNAs. Here we show two novel formulas for calculating a new measure of allele-discrimination, named "ASP-score". The formulas and ASP-score allow for an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs.

  19. Allele Workbench: transcriptome pipeline and interactive graphics for allele-specific expression.

    PubMed

    Soderlund, Carol A; Nelson, William M; Goff, Stephen A

    2014-01-01

    Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor), where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense), and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available from https

  20. Allele Workbench: Transcriptome Pipeline and Interactive Graphics for Allele-Specific Expression

    PubMed Central

    Soderlund, Carol A.; Nelson, William M.; Goff, Stephen A.

    2014-01-01

    Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor), where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense), and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available from https

  1. Epidemiological survey of Theileria parasite infection of cattle in Northeast China by allele-specific PCR.

    PubMed

    Yu, Longzheng; Zhang, Shoufa; Liang, Wanfeng; Jin, Chunmei; Jia, Lijun; Luo, Yuzi; Li, Yan; Cao, Shinuo; Yamagishi, Junya; Nishikawa, Yoshifumi; Kawano, Suguru; Fujisaki, Kozo; Xuan, Xuenan

    2011-11-01

    An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.

  2. ACNE: a summarization method to estimate allele-specific copy numbers for Affymetrix SNP arrays

    PubMed Central

    Ortiz-Estevez, Maria; Bengtsson, Henrik; Rubio, Angel

    2010-01-01

    Motivation: Current algorithms for estimating DNA copy numbers (CNs) borrow concepts from gene expression analysis methods. However, single nucleotide polymorphism (SNP) arrays have special characteristics that, if taken into account, can improve the overall performance. For example, cross hybridization between alleles occurs in SNP probe pairs. In addition, most of the current CN methods are focused on total CNs, while it has been shown that allele-specific CNs are of paramount importance for some studies. Therefore, we have developed a summarization method that estimates high-quality allele-specific CNs. Results: The proposed method estimates the allele-specific DNA CNs for all Affymetrix SNP arrays dealing directly with the cross hybridization between probes within SNP probesets. This algorithm outperforms (or at least it performs as well as) other state-of-the-art algorithms for computing DNA CNs. It better discerns an aberration from a normal state and it also gives more precise allele-specific CNs. Availability: The method is available in the open-source R package ACNE, which also includes an add on to the aroma.affymetrix framework (http://www.aroma-project.org/). Contact: arubio@ceit.es Supplementaruy information: Supplementary data are available at Bioinformatics online. PMID:20529889

  3. Allele-specific PCR for the beta-tubulin codon 200 TTC/TAC polymorphism using single adult and larval small strongyle (Cyathostominae) stages.

    PubMed

    von Samson-Himmelstjerna, G; Pape, M; von Witzendorff, C; Schnieder, T

    2002-04-01

    It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in beta-tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the beta-tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3' nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, and Cyathostomum coronatum. PCR with DNA isolated from single larvae also reproducibly generated specific fragments. This method might be applied for the future assessment of allele frequencies in susceptible and resistant populations to further investigate the mechanism of BZ-resistance in small strongyles. PMID:12053994

  4. Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

    PubMed

    Redis, Roxana S; Vela, Luz E; Lu, Weiqin; Ferreira de Oliveira, Juliana; Ivan, Cristina; Rodriguez-Aguayo, Cristian; Adamoski, Douglas; Pasculli, Barbara; Taguchi, Ayumu; Chen, Yunyun; Fernandez, Agustin F; Valledor, Luis; Van Roosbroeck, Katrien; Chang, Samuel; Shah, Maitri; Kinnebrew, Garrett; Han, Leng; Atlasi, Yaser; Cheung, Lawrence H; Huang, Gilbert Y; Monroig, Paloma; Ramirez, Marc S; Catela Ivkovic, Tina; Van, Long; Ling, Hui; Gafà, Roberta; Kapitanovic, Sanja; Lanza, Giovanni; Bankson, James A; Huang, Peng; Lai, Stephen Y; Bast, Robert C; Rosenblum, Michael G; Radovich, Milan; Ivan, Mircea; Bartholomeusz, Geoffrey; Liang, Han; Fraga, Mario F; Widger, William R; Hanash, Samir; Berindan-Neagoe, Ioana; Lopez-Berestein, Gabriel; Ambrosio, Andre L B; Gomes Dias, Sandra M; Calin, George A

    2016-02-18

    Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA. PMID:26853146

  5. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  6. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms.

    PubMed

    Matsuda, Kazuyuki; Honda, Takayuki

    2015-01-01

    Single nucleotide alternations such as single nucleotide polymorphisms (SNPs) or single nucleotide mutations are useful genetic markers for molecular diagnosis, prognosis, drug response, and predisposition to diseases. Rapid identification of SNPs or mutations is clinically important, especially for determining drug responses and selection of molecular-targeted therapy. Here, we describe a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) by using our droplet-PCR machine (droplet-AS-PCR).

  7. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  8. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping

    PubMed Central

    Lee, Han B.; Schwab, Tanya L.; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L.; Cervera, Roberto Lopez; McNulty, Melissa S.; Bostwick, Hannah S.; Clark, Karl J.

    2016-01-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  9. Allele-Specific Deletions in Mouse Tumors Identify Fbxw7 as Germline Modifier of Tumor Susceptibility

    PubMed Central

    Perez-Losada, Jesus; Wu, Di; DelRosario, Reyno; Balmain, Allan; Mao, Jian-Hua

    2012-01-01

    Genome-wide association studies (GWAS) have been successful in finding associations between specific genetic variants and cancer susceptibility in human populations. These studies have identified a range of highly statistically significant associations between single nucleotide polymorphisms (SNPs) and susceptibility to development of a range of human tumors. However, the effect of each SNP in isolation is very small, and all of the SNPs combined only account for a relatively minor proportion of the total genetic risk (5–10%). There is therefore a major requirement for alternative routes to the discovery of genetic risk factors for cancer. We have previously shown using mouse models that chromosomal regions harboring susceptibility genes identified by linkage analysis frequently exhibit allele-specific genetic alterations in tumors. We demonstrate here that the Fbxw7 gene, a commonly mutated gene in a wide range of mouse and human cancers, shows allele-specific deletions in mouse lymphomas and skin tumors. Lymphomas from three different F1 hybrids show 100% allele-specificity in the patterns of allelic loss. Parental alleles from 129/Sv or Spretus/Gla mice are lost in tumors from F1 hybrids with C57BL/6 animals, due to the presence of a specific non-synonymous coding sequence polymorphism at the N-terminal portion of the gene. A specific genetic test of association between this SNP and lymphoma susceptibility in interspecific backcross mice showed a significant linkage (p = 0.001), but only in animals with a functional p53 gene. These data therefore identify Fbxw7 as a p53-dependent tumor susceptibility gene. Increased p53-dependent tumor susceptibility and allele-specific losses were also seen in a mouse skin model of skin tumor development. We propose that analysis of preferential allelic imbalances in tumors may provide an efficient means of uncovering genetic variants that affect mouse and human tumor susceptibility. PMID:22348067

  10. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes

    PubMed Central

    Krueger, Felix; Andrews, Simon R.

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  11. A uniform survey of allele-specific binding and expression over 1000-Genomes-Project individuals.

    PubMed

    Chen, Jieming; Rozowsky, Joel; Galeev, Timur R; Harmanci, Arif; Kitchen, Robert; Bedford, Jason; Abyzov, Alexej; Kong, Yong; Regan, Lynne; Gerstein, Mark

    2016-04-18

    Large-scale sequencing in the 1000 Genomes Project has revealed multitudes of single nucleotide variants (SNVs). Here, we provide insights into the functional effect of these variants using allele-specific behaviour. This can be assessed for an individual by mapping ChIP-seq and RNA-seq reads to a personal genome, and then measuring 'allelic imbalances' between the numbers of reads mapped to the paternal and maternal chromosomes. We annotate variants associated with allele-specific binding and expression in 382 individuals by uniformly processing 1,263 functional genomics data sets, developing approaches to reduce the heterogeneity between data sets due to overdispersion and mapping bias. Since many allelic variants are rare, aggregation across multiple individuals is necessary to identify broadly applicable 'allelic elements'. We also found SNVs for which we can anticipate allelic imbalance from the disruption of a binding motif. Our results serve as an allele-specific annotation for the 1000 Genomes variant catalogue and are distributed as an online resource (alleledb.gersteinlab.org).

  12. A uniform survey of allele-specific binding and expression over 1000-Genomes-Project individuals

    PubMed Central

    Chen, Jieming; Rozowsky, Joel; Galeev, Timur R.; Harmanci, Arif; Kitchen, Robert; Bedford, Jason; Abyzov, Alexej; Kong, Yong; Regan, Lynne; Gerstein, Mark

    2016-01-01

    Large-scale sequencing in the 1000 Genomes Project has revealed multitudes of single nucleotide variants (SNVs). Here, we provide insights into the functional effect of these variants using allele-specific behaviour. This can be assessed for an individual by mapping ChIP-seq and RNA-seq reads to a personal genome, and then measuring ‘allelic imbalances' between the numbers of reads mapped to the paternal and maternal chromosomes. We annotate variants associated with allele-specific binding and expression in 382 individuals by uniformly processing 1,263 functional genomics data sets, developing approaches to reduce the heterogeneity between data sets due to overdispersion and mapping bias. Since many allelic variants are rare, aggregation across multiple individuals is necessary to identify broadly applicable ‘allelic elements'. We also found SNVs for which we can anticipate allelic imbalance from the disruption of a binding motif. Our results serve as an allele-specific annotation for the 1000 Genomes variant catalogue and are distributed as an online resource (alleledb.gersteinlab.org). PMID:27089393

  13. S-genotype identification based on allele-specific PCR in Japanese pear

    PubMed Central

    Nashima, Kenji; Terakami, Shingo; Nishio, Sogo; Kunihisa, Miyuki; Nishitani, Chikako; Saito, Toshihiro; Yamamoto, Toshiya

    2015-01-01

    Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S1–S9 and Sk) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S1/S1–S9/S9 and S4sm/S4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs. PMID:26175617

  14. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes.

    PubMed

    Krueger, Felix; Andrews, Simon R

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  15. Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75.

    PubMed

    Liu, Ying; Snedecor, Elizabeth R; Zhang, Xu; Xu, Yanfeng; Huang, Lan; Jones, Evan C; Zhang, Lianfeng; Clark, Richard A; Roop, Dennis R; Qin, Chuan; Chen, Jiang

    2016-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes. PMID:26763422

  16. Detection of Toxoplasma gondii oocysts in different water resources by Loop Mediated Isothermal Amplification (LAMP).

    PubMed

    Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Mahmoodi, Mohammad Reza; Karanis, Panagiotis

    2013-02-01

    Human toxoplasmosis is potentially contracted due to consumption of contaminated drinking water and represents an increasing public health risk worldwide. Toxoplasma gondii oocysts can be resistant to standard disinfection processes, including UV radiation. Increased awareness of the risk of waterborne toxoplasmosis outbreaks has led to an increase in research interest in the detection of oocysts in environmental water systems. Ninety-five environmental water samples from the Lower Rhine area in Germany have been included in the study and examined for the presence of Toxoplasma. Water samples were filtered or flocculated by aluminum sulfate and purified by sucrose density gradient. DNA was then extracted, and the DNA samples were then examined by LAMP analysis. T. gondii DNA was detected in eight out of 83 (9.6%) influent and effluent samples obtained from wastewater treatment plants. All samples (n=12) from the surface, ground, raw and tap waters tested negative. The purpose of this work was to investigate the occurrence and distribution of Toxoplasma oocysts on the Lower Rhine in Germany. Our study provides evidence that the assay is a sensitive, specific, rapid and cost effective method for the detection of T. gondii and is useful for both the investigations of cases of waterborne outbreaks and for identifying the source of contamination.

  17. Rapid detection of porcine kobuvirus in feces by reverse transcription loop-mediated isothermal amplification

    PubMed Central

    2014-01-01

    Background PKV is a new emerging pathogen detected in diarrhea pigs. At present, no more detection methods were reported except RT-PCR method. this study was to develop a fast diagnostic method based on the LAMP reaction for rapid detection of PKV nucleic acid in fecal samples. Findings Two pairs of primers were designed to amplify the conservative 3D gene of PKV genome. The PKV RT-LAMP method possessed well specificity and had 100 times higher sensitivity than common reverse transcription PCR (RT-PCR), which could detect up to 10 RNA copies of the target gene. Conclusions The results showed that the optimal reaction condition for RT-LAMP was achieved at 64°C for 50 min. Furthermore, the RT-LAMP procedure does not demand special equipment and is time-saving. PMID:24755372

  18. Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay.

    PubMed

    Kang, Sung-Il; Her, Moon; Kim, Ji-Yeon; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Jung, Suk Chan

    2015-06-01

    A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/μl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field.

  19. Development of loop-mediated isothermal amplification for detection of Leifsonia xyli subsp. xyli in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is prevalent in most sugarcane-producing countries. Because the disease does not cause characteristic external symptoms, a laboratory-based technique is needed for accurate diagnosis. We developed a diag...

  20. Development of loop-mediated isothermal amplification (LAMP) for detection of Theileria equi.

    PubMed

    Xie, Junren; Liu, Guangyuan; Tian, Zhancheng; Luo, Jin

    2013-09-01

    Several approaches have been developed for diagnosis of Theileria equi infection in horses and donkeys but all of them have limitations in practice. Due to numerous strengths including easy operation, cheapness and high sensitivity and specificity, LAMP has been already extensively used for surveillance of a number of diseases. We here set up a LAMP assay based on 18S rRNA gene for T. equi diagnosis. The approach was specific enough to differentiate T. equi from other evolutionary-related protozoa. Moreover, it was sensitive enough that LAMP was capable of detecting as much low as 10 copy target gene and 1 pg/μl blood genomic DNA. It was further demonstrated that LAMP was much more sensitive than canonical blood smear and comparable to PCR using test and field samples. The present results support an idea that LAMP developed in this study is reliable, reproducible and highly sensitive and specific, being a potential to be globally used for surveillance of T. equi infection in the field.

  1. Sensitive and rapid detection of genetic modified soybean (Roundup Ready) by loop-mediated isothermal amplification.

    PubMed

    Liu, Mei; Luo, Yan; Tao, Ran; He, Ru; Jiang, Keyong; Wang, Baojie; Wang, Lei

    2009-11-01

    Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening.

  2. Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus

    PubMed Central

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes. PMID:27465215

  3. Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

    PubMed Central

    Scholefield, Janine; Watson, Lauren; Smith, Danielle; Greenberg, Jacquie; Wood, Matthew JA

    2014-01-01

    Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases. PMID:24667781

  4. Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts.

    PubMed

    Scholefield, Janine; Watson, Lauren; Smith, Danielle; Greenberg, Jacquie; Wood, Matthew J A

    2014-12-01

    Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.

  5. Genome-wide survey of allele-specific splicing in humans

    PubMed Central

    Nembaware, Victoria; Lupindo, Bukiwe; Schouest, Katherine; Spillane, Charles; Scheffler, Konrad; Seoighe, Cathal

    2008-01-01

    Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST) and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array data, including several

  6. Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

    PubMed

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  7. Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

    PubMed Central

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  8. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

    PubMed

    Luo, Zhuojuan; Lin, Chengqi; Woodfin, Ashley R; Bartom, Elizabeth T; Gao, Xin; Smith, Edwin R; Shilatifard, Ali

    2016-01-01

    Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer. PMID:26728555

  9. In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides

    PubMed Central

    Southwell, Amber L; Skotte, Niels H; Kordasiewicz, Holly B; Østergaard, Michael E; Watt, Andrew T; Carroll, Jeffrey B; Doty, Crystal N; Villanueva, Erika B; Petoukhov, Eugenia; Vaid, Kuljeet; Xie, Yuanyun; Freier, Susan M; Swayze, Eric E; Seth, Punit P; Bennett, Clarence Frank; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD. PMID:25101598

  10. Microarrays for high-throughput genotyping of MICA alleles using allele-specific primer extension.

    PubMed

    Baek, I C; Jang, J-P; Choi, H-B; Choi, E-J; Ko, W-Y; Kim, T-G

    2013-10-01

    The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.

  11. Allele-specific RNAi Mitigates Phenotypic Progression in a Transgenic Model of Alzheimer's Disease

    PubMed Central

    Rodríguez-Lebrón, Edgardo; Gouvion, Cynthia M; Moore, Steven A; Davidson, Beverly L; Paulson, Henry L

    2009-01-01

    Despite recent advances suggesting new therapeutic targets, Alzheimer's disease (AD) remains incurable. Aberrant production and accumulation of the Aβ peptide resulting from altered processing of the amyloid precursor protein (APP) is central to the pathogenesis of disease, particularly in dominantly inherited forms of AD. Thus, modulating the production of APP is a potential route to effective AD therapy. Here, we describe the successful use of an allele-specific RNA interference (RNAi) approach targeting the Swedish variant of APP (APPsw) in a transgenic mouse model of AD. Using recombinant adeno-associated virus (rAAV), we delivered an anti-APPsw short-hairpin RNA (shRNA) to the hippocampus of AD transgenic mice (APP/PS1). In short- and long-term transduction experiments, reduced levels of APPsw transprotein were observed throughout targeted regions of the hippocampus while levels of wild-type murine APP remained unaltered. Moreover, intracellular production of transfer RNA (tRNA)-valine promoter–driven shRNAs did not lead to detectable neuronal toxicity. Finally, long-term bilateral hippocampal expression of anti-APPsw shRNA mitigated abnormal behaviors in this mouse model of AD. The difference in phenotype progression was associated with reduced levels of soluble Aβ but not with a reduced number of amyloid plaques. Our results support the development of allele-specific RNAi strategies to treat familial AD and other dominantly inherited neurodegenerative diseases. PMID:19532137

  12. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity

    PubMed Central

    Luo, Zhuojuan; Lin, Chengqi; Woodfin, Ashley R.; Bartom, Elizabeth T.; Gao, Xin; Smith, Edwin R.; Shilatifard, Ali

    2016-01-01

    Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer. PMID:26728555

  13. Allele-Specific Interactions between CAST AWAY and NEVERSHED Control Abscission in Arabidopsis Flowers

    PubMed Central

    Groner, William D.; Christy, Megan E.; Kreiner, Catherine M.; Liljegren, Sarah J.

    2016-01-01

    An advantage of analyzing abscission in genetically tractable model plants is the ability to make use of classic genetic tools such as suppression analysis. We have investigated the regulation of organ abscission by carrying out suppression analysis in Arabidopsis flowers. Plants carrying mutations in the NEVERSHED (NEV) gene, which encodes an ADP-ribosylation factor GTPase-activating protein, retain their outer floral organs after fertilization. Mutant alleles of CAST AWAY (CST), which encodes a receptor-like cytoplasmic kinase, were found to restore organ abscission in nev flowers in an allele-specific manner. To further explore the basis of the interactions between CST and NEV, we tested whether the site of a nev mutation is predictive of its ability to be suppressed. Our results suggest instead that the strength of a nev allele influences whether organ abscission can be rescued by a specific allele of CST.

  14. Loss of RNA expression and allele-specific expression associated with congenital heart disease

    PubMed Central

    McKean, David M.; Homsy, Jason; Wakimoto, Hiroko; Patel, Neil; Gorham, Joshua; DePalma, Steven R.; Ware, James S.; Zaidi, Samir; Ma, Wenji; Patel, Nihir; Lifton, Richard P.; Chung, Wendy K.; Kim, Richard; Shen, Yufeng; Brueckner, Martina; Goldmuntz, Elizabeth; Sharp, Andrew J.; Seidman, Christine E.; Gelb, Bruce D.; Seidman, J. G.

    2016-01-01

    Congenital heart disease (CHD), a prevalent birth defect occurring in 1% of newborns, likely results from aberrant expression of cardiac developmental genes. Mutations in a variety of cardiac transcription factors, developmental signalling molecules and molecules that modify chromatin cause at least 20% of disease, but most CHD remains unexplained. We employ RNAseq analyses to assess allele-specific expression (ASE) and biallelic loss-of-expression (LOE) in 172 tissue samples from 144 surgically repaired CHD subjects. Here we show that only 5% of known imprinted genes with paternal allele silencing are monoallelic versus 56% with paternal allele expression—this cardiac-specific phenomenon seems unrelated to CHD. Further, compared with control subjects, CHD subjects have a significant burden of both LOE genes and ASE events associated with altered gene expression. These studies identify FGFBP2, LBH, RBFOX2, SGSM1 and ZBTB16 as candidate CHD genes because of significantly altered transcriptional expression. PMID:27670201

  15. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients.

    PubMed

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-12-22

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients' samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAF(V600E) and BRAF(V600K) mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method.

  16. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

    PubMed Central

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-01-01

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

  17. Allele-specific analysis of DNA replication origins in mammalian cells.

    PubMed

    Bartholdy, Boris; Mukhopadhyay, Rituparna; Lajugie, Julien; Aladjem, Mirit I; Bouhassira, Eric E

    2015-05-19

    The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of G-quadruplex-forming sequences does not correlate with the efficiency of initiation. Sequence analysis reveals that the origins are highly enriched in sequences with profoundly asymmetric G/C and A/T nucleotide distributions and are almost completely depleted of antiparallel triplex-forming sequences. We therefore propose that although G4-forming sequences are abundant in replication origins, an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4, is directly associated with origin activity.

  18. Allele-Specific Methylation Occurs at Genetic Variants Associated with Complex Disease

    PubMed Central

    Hutchinson, John N.; Raj, Towfique; Fagerness, Jes; Stahl, Eli; Viloria, Fernando T.; Gimelbrant, Alexander; Seddon, Johanna; Daly, Mark; Chess, Andrew; Plenge, Robert

    2014-01-01

    We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results. PMID:24911414

  19. Allele-specific deposition of macroH2A1 in Imprinting Control Regions

    SciTech Connect

    Choo, J H; Kim, J D; Chung, J H; Stubbs, L; Kim, J

    2006-01-13

    In the current study, we analyzed the deposition patterns of macroH2A1 at a number of different genomic loci located in X chromosome and autosomes. MacroH2A1 is preferentially deposited at methylated CpG CpG-rich regions located close to promoters. The macroH2A1 deposition patterns at the methylated CpG islands of several imprinted domains, including the Imprinting Control Regions (ICRs) of Xist, Peg3, H19/Igf2 Igf2, Gtl2/Dlk1, and Gnas domains, show consistent allele-specificity towards inactive, methylated alleles. The macroH2A1 deposition levels at the ICRs and other Differentially Methylated Regions (DMRs) of these domains are also either higher or comparable to those observed at the inactive X chromosome of female mammals. Overall, our results indicate that besides DNA methylation macroH2A1 is another epigenetic component in the chromatin of ICRs displaying differential association with two parental alleles.

  20. Allele-specific methylation occurs at genetic variants associated with complex disease.

    PubMed

    Hutchinson, John N; Raj, Towfique; Fagerness, Jes; Stahl, Eli; Viloria, Fernando T; Gimelbrant, Alexander; Seddon, Johanna; Daly, Mark; Chess, Andrew; Plenge, Robert

    2014-01-01

    We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results.

  1. The allele-specific suppressor sup-39 alters use of cryptic splice sites in Caenorhabditis elegans.

    PubMed Central

    Roller, A B; Hoffman, D C; Zahler, A M

    2000-01-01

    Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5' end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5' splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors. PMID:10757761

  2. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    PubMed

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

  3. Assessing allele-specific expression across multiple tissues from RNA-seq read data

    PubMed Central

    Pirinen, Matti; Lappalainen, Tuuli; Zaitlen, Noah A.; Dermitzakis, Emmanouil T.; Donnelly, Peter; McCarthy, Mark I.; Rivas, Manuel A.

    2015-01-01

    Motivation: RNA sequencing enables allele-specific expression (ASE) studies that complement standard genotype expression studies for common variants and, importantly, also allow measuring the regulatory impact of rare variants. The Genotype-Tissue Expression (GTEx) project is collecting RNA-seq data on multiple tissues of a same set of individuals and novel methods are required for the analysis of these data. Results: We present a statistical method to compare different patterns of ASE across tissues and to classify genetic variants according to their impact on the tissue-wide expression profile. We focus on strong ASE effects that we are expecting to see for protein-truncating variants, but our method can also be adjusted for other types of ASE effects. We illustrate the method with a real data example on a tissue-wide expression profile of a variant causal for lipoid proteinosis, and with a simulation study to assess our method more generally. Availability and implementation: http://www.well.ox.ac.uk/~rivas/mamba/. R-sources and data examples http://www.iki.fi/mpirinen/ Contact: matti.pirinen@helsinki.fi or rivas@well.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25819081

  4. Extensive allele-specific translational regulation in hybrid mice

    PubMed Central

    Hou, Jingyi; Wang, Xi; McShane, Erik; Zauber, Henrik; Sun, Wei; Selbach, Matthias; Chen, Wei

    2015-01-01

    Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression. PMID:26253569

  5. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions.

    PubMed

    Klengel, Torsten; Mehta, Divya; Anacker, Christoph; Rex-Haffner, Monika; Pruessner, Jens C; Pariante, Carmine M; Pace, Thaddeus W W; Mercer, Kristina B; Mayberg, Helen S; Bradley, Bekh; Nemeroff, Charles B; Holsboer, Florian; Heim, Christine M; Ressler, Kerry J; Rein, Theo; Binder, Elisabeth B

    2013-01-01

    Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma-dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders. PMID:23201972

  6. Efficient and allele-specific genome editing of disease loci in human iPSCs.

    PubMed

    Smith, Cory; Abalde-Atristain, Leire; He, Chaoxia; Brodsky, Brett R; Braunstein, Evan M; Chaudhari, Pooja; Jang, Yoon-Young; Cheng, Linzhao; Ye, Zhaohui

    2015-03-01

    Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

  7. Characterization and machine learning prediction of allele-specific DNA methylation.

    PubMed

    He, Jianlin; Sun, Ming-an; Wang, Zhong; Wang, Qianfei; Li, Qing; Xie, Hehuang

    2015-12-01

    A large collection of Single Nucleotide Polymorphisms (SNPs) has been identified in the human genome. Currently, the epigenetic influences of SNPs on their neighboring CpG sites remain elusive. A growing body of evidence suggests that locus-specific information, including genomic features and local epigenetic state, may play important roles in the epigenetic readout of SNPs. In this study, we made use of mouse methylomes with known SNPs to develop statistical models for the prediction of SNP associated allele-specific DNA methylation (ASM). ASM has been classified into parent-of-origin dependent ASM (P-ASM) and sequence-dependent ASM (S-ASM), which comprises scattered-S-ASM (sS-ASM) and clustered-S-ASM (cS-ASM). We found that P-ASM and cS-ASM CpG sites are both enriched in CpG rich regions, promoters and exons, while sS-ASM CpG sites are enriched in simple repeat and regions with high frequent SNP occurrence. Using Lasso-grouped Logistic Regression (LGLR), we selected 21 out of 282 genomic and methylation related features that are powerful in distinguishing cS-ASM CpG sites and trained the classifiers with machine learning techniques. Based on 5-fold cross-validation, the logistic regression classifier was found to be the best for cS-ASM prediction with an ACC of 0.77, an AUC of 0.84 and an MCC of 0.54. Lastly, we applied the logistic regression classifier on human brain methylome and predicted 608 genes associated with cS-ASM. Gene ontology term enrichment analysis indicated that these cS-ASM associated genes are significantly enriched in the category coding for transcripts with alternative splicing forms. In summary, this study provided an analytical procedure for cS-ASM prediction and shed new light on the understanding of different types of ASM events.

  8. Allele-specific H3K79 Di- versus trimethylation distinguishes opposite parental alleles at imprinted regions.

    PubMed

    Singh, Purnima; Han, Li; Rivas, Guillermo E; Lee, Dong-Hoon; Nicholson, Thomas B; Larson, Garrett P; Chen, Taiping; Szabó, Piroska E

    2010-06-01

    Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the "histone code" at imprinted genes.

  9. Extracellular Superoxide Dismutase Polymorphism in Mice: Allele- Specific Effects on Phenotype

    PubMed Central

    Jun, Sujung; Pierce, Anson; Dory, Ladislav

    2010-01-01

    Extracellular superoxide dismutase (ecSOD) protects the extracellular matrix (ECM) from oxidative stress. We previously reported a new allele for ecSOD, expressed in 129P3/J mice (129), which differs from the wild-type (wt), expressed in C57BL/6J and other strains, by two amino acid substitutions and a 10 bp deletion in the 3' UTR of the mRNA [1]. The newly discovered allele is associated with a phenotype of significantly increased circulating and heparin-releasable enzyme activities and levels. In order to examine the properties of the two forms of ecSOD in an identical environment we generated, by extensive backcrossing of ecSOD heterozygous progeny to C57BL/6J females, a congenic C57 strain with the 129 (or wt) allele of ecSOD. These mice are homozygous for nearly 5,000 SNPs across all chromosomes, as determined by Affymetrix Parallele Mouse 5K SNP panel. The present study describes the generation of the congenic mice (genetically >99.8 % identical) and their ecSOD phenotype. The congenic mice plasma ecSOD activities before and after heparin administration recapitulate the differences reported in the founder mice. Tissue enzyme distribution is similar in both congenic groups, although the 129 allele is associated with higher levels of enzyme expression despite lower levels of enzyme mRNA. In these characteristics the phenotype is also allele driven, with little impact by the rest of the genome. The congenic mice carrying the 129 allele have mRNA levels that are in between those found in the founder 129P3/J and C57BL/6J strains. We conclude that the ecSOD phenotype in most aspects of enzyme expression is allele- driven, with the exception of tissue mRNA levels, where a significant contribution by the surrounding (host) genome is observed. These results also suggest potential allele-specific differences in the regulation of ecSOD synthesis and intracellular processing/secretion of ecSOD, independent of the genotype context. Most importantly, the congenic mice

  10. Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

    PubMed Central

    de los Monteros, Luz Elena Espinosa; Galán, Juan Carlos; Gutiérrez, Montserrat; Samper, Sofía; García Marín, Juan F.; Martín, Carlos; Domínguez, Lucas; de Rafael, Luis; Baquero, Fernando; Gómez-Mampaso, Enrique; Blázquez, Jesús

    1998-01-01

    An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform. PMID:9431955

  11. From genes to phenotypes - evaluation of two methods for the SNP analysis in archaeological remains: pyrosequencing and competitive allele specific PCR (KASPar).

    PubMed

    Pruvost, Melanie; Reissmann, Monika; Benecke, Norbert; Ludwig, Arne

    2012-01-20

    The amplification length of the DNA fragments is one major limitation of most paleogenetic analyses. Routinely, only fragments below 200 bp can be amplified, significantly reducing the content of genetic information. Although overlapping PCR strategies and next generation sequencing techniques have strongly improved data mining recently, these methods are still expensive and time consuming. In contrast, SNP analyses are easy to handle, fast and cheap. In this study, we compare two methods of SNP detection as to efficiency, cost and reliability for their use in ancient DNA applications: pyrosequencing and competitive allele specific PCR (KASPar). Our sample set consisted of 16 horse bones from two Scythian graves (600-800 BC). In conclusion, both approaches produced reliable results for most allelic patterns. But an indel of 11 bp (ASIP) could not be detected in the KASPar approach and produced problems in the pyrosequencing method (70% success rate). In such cases, we recommend checking allelic distribution using a gel approach or capillary sequencing. Overall, in comparison with the traditional mode of ancient DNA investigations (PCR, cloning, capillary sequencing), both approaches are superior for SNP analyses especially of large sample sets.

  12. Survey of benign Theileria parasites of cattle and buffaloes in Thailand using allele-specific polymerase chain reaction of major piroplasm surface protein gene.

    PubMed

    Sarataphan, Nopporn; Kakuda, Tsutomu; Chansiri, Kosum; Onuma, Misao

    2003-01-01

    During a year from 1999 to 2000, a total of 247 blood samples were collected from 214 cattle and 33 water buffaloes in 16 distinct geographical locations of Thailand and analyzed by allele-specific PCR amplification of major piroplasm surface protein (MPSP) genes of benign Theileria parasites. Four allelic MPSP gene types were determined namely C-type, I-type, B-type and Thai-type, which were originally designated from Japanese Theileria orientalis (Chitose, Ikeda), Australian T. buffeli (Warwick) and Thai T. sp. (Kamphaeng Saen), respectively. Only two allelic MPSP gene types were successively amplified from 204 (82.6%) blood samples. Among positive cases, 138 (67.6%) and 17 (8.3%) samples contained either Thai-type or C-type parasites, respectively, while 49 (24%) samples contained both types. However, nucleotide sequences of MPSP genes of Thai T. sp. amplified by C-type specific primers revealed higher (96.3%) similarity to Indonesian T. sp. rather than (87.8% similarity) to Japanese T. orientalis (Chitose) designated as C-type.

  13. Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

    PubMed

    Iliadi, Alexandra; Petropoulou, Margarita; Ioannou, Penelope C; Christopoulos, Theodore K; Anagnostopoulos, Nikolaos I; Kanavakis, Emmanuel; Traeger-Synodinos, Jan

    2011-09-01

    In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

  14. A modified approach to identification of the sickle cell anemia mutation by means of allele-specific polymerase chain reaction.

    PubMed

    Birikh, K R; Plutalov, O V; Schwartz, E I; Devi, P S; Berlin, Y A

    1992-01-01

    The allele-specific PCR approach has been modified by introducing a second mismatch at the 3'-penultimate link of the primer and used to identify the sickle cell anemia mutation (A-->T transversion in the sixth codon of the human beta-globin gene causing Glu-->Val substitution in the protein), thus obviating the problem of an interpretationally ambiguous 3'-terminal mismatch including T residue. PMID:1301951

  15. A modified approach to identification of the sickle cell anemia mutation by means of allele-specific polymerase chain reaction.

    PubMed

    Birikh, K R; Plutalov, O V; Schwartz, E I; Devi, P S; Berlin, Y A

    1992-01-01

    The allele-specific PCR approach has been modified by introducing a second mismatch at the 3'-penultimate link of the primer and used to identify the sickle cell anemia mutation (A-->T transversion in the sixth codon of the human beta-globin gene causing Glu-->Val substitution in the protein), thus obviating the problem of an interpretationally ambiguous 3'-terminal mismatch including T residue.

  16. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    PubMed

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis. PMID:25591497

  17. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    PubMed

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

  18. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi

    PubMed Central

    Kaulich, Manuel; Lee, Yeon J.; Lönn, Peter; Springer, Aaron D.; Meade, Bryan R.; Dowdy, Steven F.

    2015-01-01

    Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. PMID:25586224

  19. Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles.

    PubMed

    Ohnishi, Yusuke; Tokunaga, Katsushi; Kaneko, Kiyotoshi; Hohjoh, Hirohiko

    2006-02-28

    Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASPRNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer's disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid beta 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

  20. Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression.

    PubMed

    Cavalli, Marco; Pan, Gang; Nord, Helena; Wallerman, Ola; Wallén Arzt, Emelie; Berggren, Olof; Elvers, Ingegerd; Eloranta, Maija-Leena; Rönnblom, Lars; Lindblad Toh, Kerstin; Wadelius, Claes

    2016-05-01

    Genome-wide association studies (GWAS) have identified a large number of disease-associated SNPs, but in few cases the functional variant and the gene it controls have been identified. To systematically identify candidate regulatory variants, we sequenced ENCODE cell lines and used public ChIP-seq data to look for transcription factors binding preferentially to one allele. We found 9962 candidate regulatory SNPs, of which 16 % were rare and showed evidence of larger functional effect than common ones. Functionally rare variants may explain divergent GWAS results between populations and are candidates for a partial explanation of the missing heritability. The majority of allele-specific variants (96 %) were specific to a cell type. Furthermore, by examining GWAS loci we found >400 allele-specific candidate SNPs, 141 of which were highly relevant in our cell types. Functionally validated SNPs support identification of an SNP in SYNGR1 which may expose to the risk of rheumatoid arthritis and primary biliary cirrhosis, as well as an SNP in the last intron of COG6 exposing to the risk of psoriasis. We propose that by repeating the ChIP-seq experiments of 20 selected transcription factors in three to ten people, the most common polymorphisms can be interrogated for allele-specific binding. Our strategy may help to remove the current bottleneck in functional annotation of the genome. PMID:26993500

  1. Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia

    PubMed Central

    Wang, Xu; Clark, Andrew G.

    2016-01-01

    Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia. PMID

  2. Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia.

    PubMed

    Wang, Xu; Werren, John H; Clark, Andrew G

    2016-07-01

    Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia. PMID

  3. Identification of self-incompatibility genotypes of apricot (Prunus armeniaca L.) by S-allele-specific PCR analysis.

    PubMed

    Jie, Qi; Shupeng, Gai; Jixiang, Zhang; Manru, Gu; Huairui, Shu

    2005-08-01

    A cDNA of 417 bp encoding an S-RNase gene, named PA S3, was isolated from apricot, Prunus aremeniaca. Nine S-alleles, S1-S9, were recognized by S-allele-specific PCR and confirmed by Southern blot analysis using PA S3 as probe. The S-genotypes of the six cultivars were determined and the results of self- and cross-pollination tests among the six cultivars were consistent with the predicted S-haplotypes by PCR analysis.

  4. Pepino mosaic virus genotype shift in North America and rapid genotype identification using loop-mediated isothermal amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pepino mosaic, once an emerging disease a decade ago, has become endemic on greenhouse tomatoes worldwide in recent years. Three distinct genotypes of Pepino mosaic virus (PepMV), including EU, US1 and CH2 have been recognized. Our earlier study in 2006-2007 demonstrated a predominant EU genotype ...

  5. Detection of Puccinia kuehnii causing sugarcane orange rust with a loop-mediated isothermal amplification-based assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries including Florida and Louisiana in the U.S. At the early stage of the disease, it is almost indistinguishable from brown rust, which is caused by another species of Puccinia, viz., P...

  6. R-loop-mediated genomic instability is caused by impairment of replication fork progression.

    PubMed

    Gan, Wenjian; Guan, Zhishuang; Liu, Jie; Gui, Ting; Shen, Keng; Manley, James L; Li, Xialu

    2011-10-01

    Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.

  7. Allele-Specific Reduction of the Mutant Huntingtin Allele Using Transcription Activator-Like Effectors in Human Huntington's Disease Fibroblasts.

    PubMed

    Fink, Kyle D; Deng, Peter; Gutierrez, Josh; Anderson, Joseph S; Torrest, Audrey; Komarla, Anvita; Kalomoiris, Stefanos; Cary, Whitne