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Sample records for multiplex pcr methods

  1. A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis.

    PubMed

    Xu, Wentao; Zhai, Zhifang; Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

    2012-01-01

    In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5'-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on.

  2. Identification of Malassezia species from pityriasis versicolor lesions with a new multiplex PCR method.

    PubMed

    Vuran, Emre; Karaarslan, Aydın; Karasartova, Djursun; Turegun, Buse; Sahin, Fikret

    2014-02-01

    Despite the fact that a range of molecular methods have been developed as tools for the diagnosis of Malassezia species, there are several drawbacks associated with them, such as inefficiency of differentiating all the species, high cost, and questionable reproducibility. In addition, most of the molecular methods require cultivation to enhance sensitivity. Therefore, alternative methods eliminating cultivation and capable of identifying species with high accuracy and reliability are needed. Herein, a multiplex polymerase chain reaction (PCR)-based method was especially developed for the detection of eleven Malassezia species. The multiplex PCR was standardized by incorporating a consensus forward primer, along with Malassezia species-specific reverse primers considering the sizes of the PCR products. In the method, the multiplex-PCR primer content is divided into three parts to circumvent the problem of increased nonspecific background resulting from the use of a large number of primers. DNA extraction protocol described by Harju and colleagues was modified using liquid nitrogen instead of -80 °C to break down the yeast membrane. By a modified extraction procedure followed by multiplex PCR and electrophoresis, the method enables identification and differentiation of Malassezia species from both of the samples obtained directly from skin and yeast colonies grown in culture. Fifty-five patients who were confirmed with pityriasis versicolor were enrolled in the study. Multiplex PCR detected and differentiated all 55 samples obtained directly from the patients' skin. However, 50 out of 55 samples yielded Malassezia colony in the culture. In addition, eight of 50 colonies were misdiagnosed or not completely differentiated by conventional methods based on the sequence analysis of eight colonies. The method is capable of identifying species with high accuracy and reliability. In addition, it is simple, quick, and cost-effective. More importantly, the method works

  3. Quantitation of DNA sequences in environmental PCR products by a multiplexed, bead-based method.

    PubMed

    Spiro, Alexander; Lowe, Mary

    2002-02-01

    A first application of a multiplexed, bead-based method is described for determining the abundances of target sequences in an environmental PCR product. Target sequences as little as 0.3% of the total amount of DNA can be quantified. Tests were conducted on 16S ribosomal DNA sequences from microorganisms collected from contaminated groundwater. PMID:11823255

  4. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    PubMed

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future.

  5. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    PubMed

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future. PMID:25455748

  6. New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

    PubMed

    Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

  7. New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

    PubMed

    Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products. PMID:26257724

  8. New multiplex PCR methods for rapid screening of genetically modified organisms in foods

    PubMed Central

    Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products. PMID:26257724

  9. Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses

    PubMed Central

    Kilpatrick, David R.; Ching, Karen; Iber, Jane; Campagnoli, Ray; Freeman, Christopher J.; Mishrik, Nada; Liu, Hong-Mei; Pallansch, Mark A.; Kew, Olen M.

    2004-01-01

    The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR. PMID:15365031

  10. A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

    PubMed

    Matsuoka, T; Kuribara, H; Akiyama, H; Miura, H; Goda, Y; Kusakabe, Y; Isshiki, K; Toyoda, M; Hino, A

    2001-02-01

    Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

  11. Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses.

    PubMed

    Gambino, Giorgio

    2015-01-01

    Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples.

  12. Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

    PubMed Central

    Alam, Mohammad J.; Tisdel, Naradah L.; Shah, Dhara N.; Yapar, Mehmet; Lasco, Todd M.; Garey, Kevin W.

    2015-01-01

    Background The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. Conclusions The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. PMID:25932438

  13. Multiplex-PCR Method for Species Identification of Coagulase-Positive Staphylococci ▿

    PubMed Central

    Sasaki, Takashi; Tsubakishita, Sae; Tanaka, Yoshikazu; Sakusabe, Arihito; Ohtsuka, Masayuki; Hirotaki, Shintaro; Kawakami, Tetsuji; Fukata, Tsuneo; Hiramatsu, Keiichi

    2010-01-01

    In veterinary medicine, coagulase-positive staphylococci (CoPS) other than Staphylococcus aureus have frequently been misidentified as being S. aureus strains, as they have several phenotypic traits in common. There has been no reliable method to distinguish among CoPS species in veterinary clinical laboratories. In the present study, we sequenced the thermonuclease (nuc) genes of staphylococcal species and devised a multiplex-PCR (M-PCR) method for species identification of CoPS by targeting the nuc gene locus. To evaluate sensitivity and specificity, we used this M-PCR method on 374 staphylococcal strains that had been previously identified to the species level by an hsp60 sequencing approach. We could successfully distinguish between S. aureus, S. hyicus, S. schleiferi, S. intermedius, S. pseudintermedius, and S. delphini groups A and B. The present method was both sensitive (99.8%) and specific (100%). Our M-PCR assay will allow the routine species identification of CoPS isolates from various animal species for clinical veterinary diagnosis. PMID:20053855

  14. Development of a rapid identification method for Aeromonas species by multiplex-PCR.

    PubMed

    Sen, Keya

    2005-11-01

    Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.

  15. Molecular identification of Cycospora spp. using multiplex PCR from diarrheic children compared to others conventional methods.

    PubMed

    Hussein, Eman M

    2007-08-01

    Cyclospora organisms named C. cercopitheci, C. colobi & C. papionis were identified in stool samples from several primates. They were morphologically indistinguishable from C. cayetanensis but genetically different. In the present work, Cyclospora infection was diagnosed among 140 diarrheic children with three conventional diagnostic methods and was confirmed using nested PCR. The possibility of infection with not only C. cayetanensis but the three Cyclospora species of primates was identified by multiplex PCR among all cyclosporiasis patients diagnosed by different methods. The results showed that Cyclospora was detected in 25 (17.8%), 31 (22.2%), 32 (22.9%) & 35 (25%) patients using modified Kinyoun stain, auto-fluorescent characteristics, sporulation process of the oocysts and nested PCR respectively. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value and Kappa test were calculated in relation to nested PCR. The single nucleotide polymorphisms (SNPs) in the 18S rRNA gene of C. cayetanensis were identified in 35 cyclosporiasis patients and only one patient had the possibility of human coinfection with primates Cyclospora species (C. cercopitheci, C. colobi & C. papionis) and C. cayetanensis by appearance of a 361-bp. Scanning electron microscopy proved no morphological differences could be detected among Cyclospora oocysts isolated from this patient.

  16. Multiplex PCR method to discriminate Artemisia iwayomogi from other Artemisia plants.

    PubMed

    Doh, Eui Jeong; Oh, Seung-Eun

    2012-01-01

    Some plants in the genus Artemisia have been used for medicinal purposes. Among them, Artemisia iwayomogi, commonly referred to as "Haninjin," is one of the major medicinal materials used in traditional Korean medicine. By contrast, Artemisia capillaris and both Artemisia argyi and Artemisia princeps, referred to as "Injinho" and "Aeyup," respectively, are used to treat diseases different from those for which "Haninjin" is prescribed. Therefore, the development of a reliable method to differentiate each Artemisia herb is necessary. We found that a random amplified polymorphic DNA (RAPD) method can be used to efficiently discriminate a few Artemisia plants from one another. To improve the reliability of RAPD amplification, we designed primer sets based on the nucleotide sequences of RAPD products to amplify a sequence-characterized amplified region (SCAR) marker of A. iwayomogi. In addition, we designed two other primer sets to amplify SCAR markers of "Aeyup" (A. argyi and A. princeps) along with "Injinho" (A. capillaris) and Artemisia japonica, which are also traded in Korean herbal markets. Using these three primer sets, we developed a multiplex PCR method concurrently not only to discriminate A. iwayomogi from other Artemisia plants, but also to identify Artemisia plants using a single PCR process.

  17. Analysis of RHD genes in Taiwanese RhD-negative donors by the multiplex PCR method.

    PubMed

    Lee, Y-L; Chiou, H-L; Hu, S-N; Wang, L

    2003-01-01

    The determination of the RhD phenotype is important in transfusion medicine. However, due to the complexity of D antigen expression, the routine serological method cannot differentiate all RhD variants. In addition, the induction of the anti-D antibody is still the major cause of severe hemolytic disease of the newborn (HDN). Therefore, it is important to understand RHD gene profiles. To analyze the RHD gene profiles of Taiwanese RhD-negative donors, the multiplex PCR method was applied to amplify RHD specific exons 3, 4, 5, 7, and 9. Based on the PCR results, the 156 RhD-negative donors were divided into 12 groups according to the different expression patterns of the RHD gene. These 12 groups were further divided into three categories: type I=Rh D(el) (21.8%); type II = partial D, containing some exons (9.0%); and type III = true RhD-negative (69.2%). The results indicated that 21.8% of RhD-negative donors in Taiwan were RhD(el), and 9% carried a part of the RHD gene. Six defined RhD variants were found in this study: four R(O) (Har), one D(Va), and two D(IVb). However, no true RhD-negative or RhD(el) donor with the CcdEe phenotype was found in this analysis.

  18. Development of a Multiplex PCR Method to Detect Fungal Pathogens for Quarantine on Exported Cacti.

    PubMed

    Cho, Hyun Ji; Hong, Seong Won; Kim, Hyun-Ju; Kwak, Youn-Sig

    2016-02-01

    Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B. cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum), 510-bp (B. cactivora), 313-bp (P. nicotinae), and 447-bp (P. cactorum). The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti. PMID:26889115

  19. Prospective Study of Congenital Toxoplasmosis Screening with Use of IgG Avidity and Multiplex Nested PCR Methods

    PubMed Central

    Yamada, Hideto; Nishikawa, Akira; Yamamoto, Tomohiro; Mizue, Yuka; Yamada, Takashi; Morizane, Mayumi; Tairaku, Shinya; Nishihira, Jun

    2011-01-01

    Acute infection with Toxoplasma gondii during pregnancy can cause congenital toxoplasmosis. The aim of this study was to evaluate whether screening with the use of IgG avidity and multiplex nested PCR methods was effective to detect a high-risk pregnancy. In a prospective study, serum T. gondii IgG avidity was measured in consecutive 146 pregnant women testing positive for T. gondii antibody and either positive or equivocal for IgM. Multiplex nested PCR for T. gondii DNA on amniotic fluid, maternal blood, and umbilical cord blood were performed with informed consent. A total of 51 (34.9%) women presented with low IgG avidity (<30%), 15 (10.3%) presented with borderline avidity (30 to 35%), and 80 (54.8%) presented with high avidity (>35%) indices. Amniotic fluid obtained at amniocentesis or birth yielded positive PCR results in nine women with low IgG avidity indices. Of these nine women, three had congenital toxoplasmosis. None of women with high or border line IgG avidity indices had a positive PCR result in the amniotic fluid or congenital toxoplasmosis. No congenital toxoplasmosis was detected in women whose amniotic fluids yielded negative PCR results. Ingestion of raw or undercooked meat was found to be the main risk factor for acute T. gondii infection. Congenital toxoplasmosis screening with a combination of IgG avidity in the maternal blood and multiplex nested PCR in the amniotic fluid was useful for detecting a high risk pregnancy and diagnosing congenital toxoplasmosis. PMID:21543572

  20. Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR.

    PubMed

    Wintermantel, William M; Hladky, Laura L

    2010-12-01

    A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and small fruit crops, and rapidly identify viruses associated with disease symptoms, as well as identification of mixed crinivirus infections. Four host groups for multiplex detection of criniviruses were selected based on the types of crops where specific criniviruses would be expected to occur. Each detection group contained three to four crop-specific primers designed to the same region of the gene encoding the highly conserved RNA-dependent RNA polymerase gene (RdRp) of criniviruses for rapid, single-reaction determination of which crinivirus(es) may be infecting a plant. Degenerate reverse primers used for RT and in PCR were designed to amplify all members of each host group, and were coupled with species-specific forward primers resulting in four separate single-reaction cocktails for detection of most criniviruses sequenced to date, whether present in single or mixed virus infections. Additional viruses can be added to multiplex detection by adjustment of primer concentration for balanced detection of target viruses. In order to identify unknown putative criniviruses or those for which sequence information is not yet available, a genus-wide, universal degenerate primer set was developed. These primers also targeted the crinivirus RdRp gene, and amplify a wide range of crinivirus sequences. Both detection systems can be used with most RNA extraction methods, and with RT-PCR reagents common in most laboratories.

  1. A New Multiplex-PCR for Urinary Tract Pathogen Detection Using Primer Design Based on an Evolutionary Computation Method.

    PubMed

    García, Liliana Torcoroma; Cristancho, Laura Maritza; Vera, Erika Patricia; Begambre, Oscar

    2015-10-01

    This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex- PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

  2. Multiplexed Primer Prediction for PCR

    SciTech Connect

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequences used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.

  3. Multiplexed Primer Prediction for PCR

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequencesmore » used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.« less

  4. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods.

    PubMed

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-13

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3' ends of cDNA is performed according to the modified classic 3' RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5' ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5' sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5' and promoter sequences. The 5' end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5' ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness.

  5. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection.

    PubMed

    Roberts, Christine C; Swoyer, Ryan; Bryan, Janine T; Taddeo, Frank J

    2011-05-01

    Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.

  6. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  7. A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius).

    PubMed

    Biswas, Chinmay; Dey, Piyali; Gotyal, B S; Satpathy, Subrata

    2015-04-01

    The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray.

  8. A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius).

    PubMed

    Biswas, Chinmay; Dey, Piyali; Gotyal, B S; Satpathy, Subrata

    2015-04-01

    The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray. PMID:25680421

  9. [Respiratory viral diagnosis by using an automated system of multiplex PCR (FilmArray) compared to conventional methods].

    PubMed

    Marcone, Débora N; Carballal, Guadalupe; Ricarte, Carmen; Echavarria, Marcela

    2015-01-01

    Acute respiratory infections, which are commonly caused by viruses, are an important cause of morbidity and mortality in children. In Argentina, national surveillance programs for the detection of respiratory viruses are usually performed by using immunofluorescence (IF) assays, although it is well known that molecular methods are more sensitive. An automated multiplex PCR device, the FilmArray-Respiratory Panel (FilmArray-RP), can detect 17 viral and 3 bacterial pathogens in a closed system that requires only 5 min of hands-on time and 1h of instrumentation time. A total of 315 respiratory samples from children under 6 years of age suffering from acute respiratory infections, were studied by IF for 8 respiratory viruses and by RT-PCR for rhinoviruses. Later, these samples were tested by the FilmArray-RP. The positivity frequency obtained for the 9 viruses tested was 75% by IF/RT-PCR and 92% by the FilmArray-RP. The positive and negative percent agreement between both methods was 70.5% whereas the negative percent agreement was 99.6% (95% confidence interval:65.5-75.1 and 99.2-99.8 respectively). The FilmArray-RP allowed a higher positive diagnosis (97%) and detected other viruses such as coronavirus NL63, 229E, OC43, HKU1 (10%) and bocavirus (18%). In addition, this method identified multiple coinfections (39%) with 2, 3, 4 and up to 5 different viruses. At present, IF is still the most frequently used method in most Latin American countries for respiratory viruses diagnosis due to its low cost, its capability to process a high number of samples simultaneously and the fast determination of results for the most frequent viruses, which are available within 5h. However, the coming incorporation of molecular methods in routine procedures will significantly increase the diagnostic yield of these infections.

  10. Detection of genetically modified microorganisms in soil using the most-probable-number method with multiplex PCR and DNA dot blot.

    PubMed

    Yeom, Jinki; Lee, Yunho; Noh, Jaemin; Jung, Jaejoon; Park, Jungsoon; Seo, Hyoju; Kim, Jisun; Han, Jiwon; Jeon, Che Ok; Kim, Taesung; Park, Woojun

    2011-10-01

    The principal objective of this study was to detect genetically modified microorganisms (GMMs) that might be accidentally released into the environment from laboratories. Two methods [plate counting and most-probable-number (MPN)] coupled with either multiplex PCR or DNA dot blots were compared using genetically modified Escherichia coli, Pseudomonas putida, and Acinetobacter oleivorans harboring an antibiotic-resistance gene with additional gfp and lacZ genes as markers. Alignments of sequences collected from databases using the Perl scripting language (Perl API) and from denaturing gradient gel electrophoresis analysis revealed that the gfp, lacZ and antibiotic-resistance genes (kanamycin, tetracycline, and ampicillin) in GMMs differed from the counterpart genes in many sequenced genomes and in soil DNA. Thus, specific multiplex PCR primer sets for detection of plasmid-based gfp and lacZ antibiotic-resistance genes could be generated. In the plate counting method, many antibiotic-resistant bacteria from a soil microcosm grew as colonies on antibiotic-containing agar plates. The multiplex PCR verification of randomly selected antibiotic-resistant colonies with specific primers proved ineffective. The MPN-multiplex PCR method and antibiotic-resistant phenotype could be successfully used to detect GMMs, although this method is quite laborious. The MPN-DNA dot blot method screened more cells at a time in a microtiter plate containing the corresponding antibiotics, and was shown to be a more efficient method for the detection of GMMs in soil using specific probes in terms of labor and accuracy. PMID:21810467

  11. Rapid Multiplex Nested PCR for Detection of Respiratory Viruses▿

    PubMed Central

    Lam, W. Y.; Yeung, Apple C. M.; Tang, Julian W.; Ip, Margaret; Chan, Edward W. C.; Hui, Mamie; Chan, Paul K. S.

    2007-01-01

    Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken. PMID:17804659

  12. An in-house multiplex pcr method to detect of putative virulence factors in aeromonas species

    PubMed Central

    Aguilera-Arreola, Ma. Guadalupe; Martínez, Alma Aidee Carmona; Castro-Escarpulli, Graciela

    2011-01-01

    A pentaplex PCR was developed and optimised to detect the genes that encode the five most important putative virulence factors in Aeromonas isolates. It seems to be more efficient than previously reported techniques and promises to be a powerful tool for more accurate risk assessments and for monitoring pathogenic strains. PMID:24031758

  13. Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

    PubMed

    Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

    2015-02-01

    Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

  14. Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

    PubMed

    Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

    2015-02-01

    Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications. PMID:25710151

  15. Development of multiplex PCR method for simultaneous detection of four events of genetically modified maize: DAS-59122-7, MIR604, MON863 and MON88017.

    PubMed

    Oguchi, Taichi; Onishi, Mari; Mano, Junichi; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Kitta, Kazumi

    2010-01-01

    A novel multiplex PCR method was developed for simultaneous event-specific detection of four events of GM maize, i.e., DAS-59122-7, MIR604, MON88017, and MON863. The single laboratory examination of analytical performance using simulated DNA mixtures containing GM DNA at various concentrations in non-GM DNA suggested that the limits of detection (LOD) of the multiplex PCR method were 0.16% for MON863, MIR604, and MON88017, and 0.078% for DAS-59122-7. We previously developed a nonaplex (9plex) PCR method for eight events of GM maize, i.e., Bt11, Bt176, GA21, MON810, MON863, NK603, T25, and TC1507. Together with the nonaplex PCR method, the newly developed method enabled the detection and identification of eleven GM maize events that are frequently included in commercial GM seed used in Japan. In addition, this combinational analysis may be useful for the identification of combined event products of GM maize. PMID:20595789

  16. Development of multiplex PCR method for simultaneous detection of four events of genetically modified maize: DAS-59122-7, MIR604, MON863 and MON88017.

    PubMed

    Oguchi, Taichi; Onishi, Mari; Mano, Junichi; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Kitta, Kazumi

    2010-01-01

    A novel multiplex PCR method was developed for simultaneous event-specific detection of four events of GM maize, i.e., DAS-59122-7, MIR604, MON88017, and MON863. The single laboratory examination of analytical performance using simulated DNA mixtures containing GM DNA at various concentrations in non-GM DNA suggested that the limits of detection (LOD) of the multiplex PCR method were 0.16% for MON863, MIR604, and MON88017, and 0.078% for DAS-59122-7. We previously developed a nonaplex (9plex) PCR method for eight events of GM maize, i.e., Bt11, Bt176, GA21, MON810, MON863, NK603, T25, and TC1507. Together with the nonaplex PCR method, the newly developed method enabled the detection and identification of eleven GM maize events that are frequently included in commercial GM seed used in Japan. In addition, this combinational analysis may be useful for the identification of combined event products of GM maize.

  17. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing.

    PubMed

    Zhang, Rui; Li, Xin; Ramaswami, Gokul; Smith, Kevin S; Turecki, Gustavo; Montgomery, Stephen B; Li, Jin Billy

    2014-01-01

    We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

  18. Development and Application of a Multiplex PCR Method for Rapid Differential Detection of Subgroup A, B, and J Avian Leukosis Viruses

    PubMed Central

    Gao, Qi; Yun, Bingling; Wang, Qi; Jiang, Lili; Zhu, Haibo; Gao, Yanni; Qin, Liting; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei

    2014-01-01

    Avian leukosis virus (ALV) subgroups A, B, and J are very common in poultry flocks and have caused serious economic losses in recent years. A multiplex PCR (mPCR) method for the detection of these three subgroups was developed and optimized in this study. We first designed a common forward primer, PF, and three downstream primers, AR, BR, and JR, which can amplify 715 bp for subgroup A, 515 bp for subgroup B, and 422 bp for subgroup J simultaneously in one reaction. The mPCR method produced neither cross-reactions with other subgroups of ALVs nor nonspecific reactions with other common avian viruses. The detection limit of the mPCR was as low as 1 × 103 viral DNA copies of each of the three subgroups. In animal experiments, the mPCR detected ALVs 2 to 4 days earlier than did virus isolation from whole-blood samples and cloaca swabs. Furthermore, a total of 346 clinical samples (including 127 tissue samples, 86 cloaca swabs, 59 albumen samples, and 74 whole-blood samples) from poultry flocks with suspected ALV infection were examined by mPCR, routine PCR, and virus isolation. The positive sample/total sample ratios for ALV-A, ALV-B, and ALV-J were 48% (166/346) as detected by mPCR and 48% (166/346) as detected by routine PCR. However, the positive sample/total sample ratio detected by virus isolation was 40% (138/346). The results of the mPCR and routine PCR were confirmed by sequencing the specific fragments. These results indicate that the mPCR method is rapid, specific, sensitive, and convenient for use in epidemiological studies of ALV, clinical detection of ALV, and ALV eradication programs. PMID:24131697

  19. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

    PubMed Central

    Stulberg, Michael J.; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum. PMID:26426354

  20. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    DOE PAGESBeta

    Stulberg, Michael J.; Huang, Qi

    2015-10-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regionsmore » of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.« less

  1. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

  2. Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

    PubMed Central

    2012-01-01

    Background Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Results Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Conclusion Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments

  3. Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

    PubMed

    Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

    2013-08-01

    Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

  4. Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

    PubMed

    Hernández, Marta; Rodríguez-Lázaro, David; Zhang, David; Esteve, Teresa; Pla, Maria; Prat, Salomé

    2005-05-01

    The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

  5. A novel multiplex RT-qPCR method based on dual-labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus.

    PubMed

    Vázquez, D; López-Vázquez, C; Skall, H F; Mikkelsen, S S; Olesen, N J; Dopazo, C P

    2016-04-01

    Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure--named binary multiplex RT-qPCR (bmRT-qPCR)--for simultaneous detection and typing of all four genotypes of VHSV by real-time RT-PCR based on dual-labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.

  6. Development of a sensitive and specific multiplex PCR method for the simultaneous detection of chicken, duck and goose DNA in meat products.

    PubMed

    Hou, Bo; Meng, Xianrong; Zhang, Liyuan; Guo, Jinyue; Li, Shaowen; Jin, Hui

    2015-03-01

    Identifying the origin of animal species in manufactured meat products is of considerable economic, religious and sanitary importance. In this study, we developed a multiplex PCR method to simultaneously detect chicken, duck and goose DNA in meat products derived from beef, pork, mutton or quail. The PCR primers were designed based on the sequence of mitochondrial genes of each avian species, and the amplicon sizes were 131, 283 and 387bp for chicken, duck and goose, respectively. The method had no cross-reaction with DNA isolated from beef, mutton, pork or quail, and generated products at a target DNA content as low as 0.05ng, or a target meat content of 1% of total meat weight. Moreover, screening of 24 commercial meat samples using this method indicated that six, two and one samples were contaminated with chicken, duck, or both, respectively, suggesting its usefulness for the simultaneous identification of chicken, duck and goose DNA in commercial meat products.

  7. A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits.

    PubMed

    Karamitros, Timokratis; Magiorkinis, Gkikas

    2015-12-15

    The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions. Oxford Nanopore Technologies recently released an USB3.0-interfaced sequencer, the MinION. To date no particular method for enriching MinION libraries has been standardized. Here, using biotinylated PCR-generated baits in a novel approach, we describe a simple and efficient way for multiplexed enrichment of MinION libraries, overcoming technical limitations related with the chemistry of the sequencing-adapters and the length of the DNA fragments. Using Phage Lambda and Escherichia coli as models we selectively enrich for specific targets, significantly increasing the corresponding read-coverage, eliminating unwanted regions. We show that by capturing genomic fragments, which contain the target sequences, we recover reads extending targeted regions and thus can be used for the determination of potentially unknown flanking sequences. By pooling enriched libraries derived from two distinct E. coli strains and analyzing them in parallel, we demonstrate the efficiency of this method in multiplexed format. Crucially we evaluated the optimal bait size for large fragment libraries and we describe for the first time a standardized method for target enrichment in MinION platform.

  8. Detection of adenoviruses and enteroviruses in tap water and river water by reverse transcription multiplex PCR.

    PubMed

    Cho, H B; Lee, S H; Cho, J C; Kim, S J

    2000-05-01

    A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to simultaneously detect adenoviruses and enteroviruses, both of which have attracted much attention as molecular indices of viral pollution in environmental samples. The method involves a reverse transcription step, followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and adenoviruses. The sensitivity of this assay was found to be similar to that of each monoplex PCR or RT-PCR assay, and to be consistent regardless of relative concentrations of adenoviruses and enteroviruses. To assess suitability and environmental application of the RT multiplex PCR assay, a total of 12 river water samples and 4 tap water samples were analyzed by RT multiplex PCR, each monoplex PCR or RT-PCR, and cell culture assay on the Buffalo Green Monkey kidney cell line. The sensitivity of the RT multiplex PCR was also found to be similar to that of each monoplex PCR in environmental samples. This suggests the RT multiplex PCR assay could be applied to the routine monitoring of viral pollution in environmental waters.

  9. Automated Methods for Multiplexed Pathogen Detection

    SciTech Connect

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However

  10. Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

    PubMed

    Zhong, Qun; Bhattacharya, Smiti; Kotsopoulos, Steven; Olson, Jeff; Taly, Valérie; Griffiths, Andrew D; Link, Darren R; Larson, Jonathan W

    2011-07-01

    Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.

  11. Identification of clinical aeromonas species by rpoB and gyrB sequencing and development of a multiplex PCR method for detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media.

    PubMed

    Persson, Søren; Al-Shuweli, Suzan; Yapici, Seval; Jensen, Joan N; Olsen, Katharina E P

    2015-02-01

    Conventional identification of Aeromonas species based on biochemical methods is challenged by the heterogeneous nature of the species. Here, we present a new multiplex PCR method directed toward the gyrB and rpoB genes that identifies four Aeromonas species, A. hydrophila, A. media, A. veronii, and A. caviae, and we describe the application of this method on a Danish strain collection.

  12. Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

    PubMed

    Noguchi, Akio; Nakamura, Kosuke; Sakata, Kozue; Sato-Fukuda, Nozomi; Ishigaki, Takumi; Mano, Junichi; Takabatake, Reona; Kitta, Kazumi; Teshima, Reiko; Kondo, Kazunari; Nishimaki-Mogami, Tomoko

    2016-04-19

    A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

  13. Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

    PubMed

    Noguchi, Akio; Nakamura, Kosuke; Sakata, Kozue; Sato-Fukuda, Nozomi; Ishigaki, Takumi; Mano, Junichi; Takabatake, Reona; Kitta, Kazumi; Teshima, Reiko; Kondo, Kazunari; Nishimaki-Mogami, Tomoko

    2016-04-19

    A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study. PMID:27010783

  14. Multiplex real-time PCR and culture methods for detection of Shiga toxin-producing Escherichia coli and Salmonella Thompson in strawberries, a lettuce mix and basil.

    PubMed

    Delbeke, S; Ceuppens, S; Holvoet, K; Samuels, E; Sampers, I; Uyttendaele, M

    2015-01-16

    An appropriate approach of high throughput multi-screening was verified for Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp. in strawberries, lettuce and basil. Sample replicates were inoculated with STEC O157 or O26 and Salmonella Thompson (ca. 10-70, 100-700 and 1000-7000 cfu/25 g) and analysed after 1 and 5 days of storage (strawberries and lettuce at 7 °C and basil at 10 °C). After 18-24 h of enrichment at 37 °C in buffered peptone water, detection was performed using the GeneDisc multiplex PCR (stx1, stx2, eae and iroB genes) and selective culture media for isolation of STEC (with immunomagnetic separation (IMS)) and Salmonella spp. in parallel. After 1 day, the pathogenic strains were recovered from all samples for all inoculum levels, whereas reduced detection rates of STEC O157 and S. Thompson were observed after 5 days of storage in case of strawberries, in particular for the lowest inoculums level, suggesting superior survival potential for STEC O26. Overall, this study indicates the ability of PCR based screening methods for reproducible multi-detection of low numbers (10-70 cfu/25 g) of STEC and Salmonella in this type of foods. However, for the basil samples, PCR needed twofold dilution of the DNA extract to overcome inhibition. It was noted that on several occasions growth of competitive microbiota obstructed finding presumptive colonies on the selective agar media, whereas the use of an additional agar medium such as CHROMagar STEC (without IMS) improved recovery rate of STEC. PMID:25462916

  15. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

    PubMed

    Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

    2004-05-20

    In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

  16. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  17. A Conventional Multiplex PCR Assay for the Detection of Toxic Gemfish Species (Ruvettus pretiosus and Lepidocybium flavobrunneum): A Simple Method To Combat Health Frauds.

    PubMed

    Giusti, Alice; Castigliego, Lorenzo; Rubino, Rossella; Gianfaldoni, Daniela; Guidi, Alessandra; Armani, Andrea

    2016-02-01

    The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been reported in sushi catering. This work developed a simple conventional multiplex PCR, which discriminates the two toxic gemfishes from other potentially replaced species, such as tunas, cod, and sablefish. A common degenerate forward primer and three species-specific reverse primers were designed to amplify cytochrome oxidase subunit I (COI) gene regions of different lengths (479, 403, and 291 bp) of gemfishes, tunas, and sablefish, respectively. A primer pair was designed to amplify a fragment (193 bp) of the cytb gene of cod species. Furthermore, a primer pair targeting the 16S rRNA gene was intended as common positive control (115 bp). The method developed in this study, by producing the expected amplicon for all of the DNA samples tested (reference and commercial), provides a rapid and reliable response in identifying the two toxic species to combat health frauds. PMID:26739756

  18. A simple procedure eliminating multiple optimization steps required in developing multiplex PCR reactions

    SciTech Connect

    Grondin, V.; Roskey, M.; Klinger, K.; Shuber, T.

    1994-09-01

    The PCR technique is one of the most powerful tools in modern molecular genetics and has achieved widespread use in the analysis of genetic diseases. Typically, a region of interest is amplified from genomic DNA or cDNA and examined by various methods of analysis for mutations or polymorphisms. In cases of small genes and transcripts, amplification of single, small regions of DNA are sufficient for analysis. However, when analyzing large genes and transcripts, multiple PCRs may be required to identify the specific mutation or polymorphism of interest. Ever since it has been shown that PCR could simultaneously amplify multiple loci in the human dystrophin gene, multiplex PCR has been established as a general technique. The properities of multiplex PCR make it a useful tool and preferable to simultaneous uniplex PCR in many instances. However, the steps for developing a multiplex PCR can be laborious, with significant difficulty in achieving equimolar amounts of several different amplicons. We have developed a simple method of primer design that has enabled us to eliminate a number of the standard optimization steps required in developing a multiplex PCR. Sequence-specific oligonucleotide pairs were synthesized for the simultaneous amplification of multiple exons within the CFTR gene. A common non-complementary 20 nucleotide sequence was attached to each primer, thus creating a mixture of primer pairs all containing a universal primer sequence. Multiplex PCR reactions were carried out containing target DNA, a mixture of several chimeric primer pairs and primers complementary to only the universal portion of the chimeric primers. Following optimization of conditions for the universal primer, limited optimization was needed for successful multiplex PCR. In contrast, significant optimization of the PCR conditions were needed when pairs of sequence specific primers were used together without the universal sequence.

  19. Multiplexing Short Primers for Viral Family PCR

    SciTech Connect

    Gardner, S N; Hiddessen, A L; Hara, C A; Williams, P L; Wagner, M; Colston, B W

    2008-06-26

    We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets for large, diverse, and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ({approx}3700 18-mers or {approx}2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus, hemagglutinin and neuraminidase segments of influenza A virus, Norwalk virus, and HIV-1.

  20. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed.

  1. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  2. [Multiplex PCR for detecting genotypes of deletional alpha-thalassemia].

    PubMed

    Wu, Jie-Ying; Liao, Can; Li, Jian; Huang, Yi-Ning

    2004-08-01

    To investigate the clinical application of multiplex PCR in detecting genotypes of deletional alpha-thalassemia in South China and observe the distribution frequency of alpha-globin gene deletion, 145 patients with silent carrier, alpha thalassemia trait or HbH were identified by M-PCR and 1.2% agarose gel electrophoresis. There are 1.3, 1.6, 1.8 and 2.0 kb bands which indicate --(SEA), -alpha(4.2), alphaalpha and -alpha(3.7), respectively. The results showed that among 145 patients, 100 patients with --(SEA)/alphaalpha (68.9%), 15 with -alpha(3.7)/alphaalpha (10.3%), 8 with -alpha(4.2)/alphaalpha (5.52%), 2 with -alpha(3.7)/-alpha(4.2) (1.38%), 1 with -alpha(3.7)/-alpha(3.7) (0.69%), 1 with -alpha(4.2)/-alpha(4.2) (0.69%), 14 with --(SEA)/-alpha(3.7) (9.65%), 2 with --(SEA)/-alpha(4.2) (1.38%) were found. Two patients prenatal diagnosed were confirmed with Bart's hydrops fetuses. In conclusion, M-PCR analysis is a simple, rapid and accurate method for detection of alpha-thalassemia gene deletion. This technique is helpful in screening, carrier identification and prenatal diagnosis of deletional alpha-thalassemia.

  3. A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray.

    PubMed

    Li, Yang; Guo, Shu-Juan; Shao, Ning; Tu, Shun; Xu, Miao; Ren, Zhao-Rui; Ling, Xing; Wang, Guo-Qing; Lin, Zhi-Xin; Tao, Sheng-Ce

    2011-11-01

    Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality

  4. Multiplex SYBR Green Real-Time PCR Assay for Detection of Respiratory Viruses

    PubMed Central

    Sultani, Mozhdeh; Mokhtari Azad, Talat; Eshragian, Mohammadreza; Shadab, Azadeh; Naseri, Maryam; Eilami, Owrang; Yavarian, Jila

    2015-01-01

    Background: It is often difficult for a physician to distinguish between viral and bacterial causes of respiratory infections and this may result in overuse of antibiotics. In many cases of community-acquired respiratory infections, clinicians treat patients empirically. The development of molecular methods for direct detection of viruses has been progressed recently. Objectives: The objective of this study was recognizing the panel of respiratory RNA viruses by multiplex SYBR Green real-time polymerase chain reaction (PCR). Materials and Methods: Randomized 172 influenza-negative respiratory specimens of all age groups of hospitalized patients were collected. After RNA extraction, cDNA was synthesized. Three SYBR Green multiplex real-time PCR assays were developed for simultaneous detection of 12 respiratory RNA viruses. Each set of multiplex methods detected four viruses, the first set: respiratory syncytial virus, human metapneumovirus, rhinovirus, enterovirus; the second set: parainfluenza viruses 1 - 4 (PIV1-4); the third set: coronaviruses NL63, 229E, severe acute respiratory syndrome (SARS), and OC43. Results: Application of the multiplex SYBR Green real-time PCR in clinical samples from 172 patients in a one-year study resulted in detection of 19 (11.04%) PIV3, 9 (5.23%) PIV4, and 1 (0.58%) coronavirus NL63. All the positive samples were detected during December to March (2011 - 2012). Conclusions: Multiplex SYBR Green real-time PCR is a rapid and relatively inexpensive method for detection of respiratory viruses. PMID:26468358

  5. Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

    PubMed

    Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

    2015-01-01

    An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences. PMID:26525256

  6. Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

    PubMed

    Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

    2015-01-01

    An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

  7. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR.

    PubMed

    Clifford, Gary M; Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A M; Snijders, Peter J F; Gheit, Tarik

    2016-08-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. PMID:27225411

  8. [Study by multiplex PCR of Listeria monocytogenes serotypes isolated in Argentine].

    PubMed

    Callejo, R; Prieto, M; Martínez, C; Aguerre, L; Rocca, F; Martínez, G; Palmieri, O

    2008-01-01

    A multiplex PCR assay, recently validated to characterize the serotypes of Listeria monocytogenes was evaluated in comparison to conventional serotyping. Three hundred forty two L. monocytogenes strains isolated from human, food, animal and environmental sources during the 1992-2005 period were assayed. The concordance between the two methods for serotypes 1/2a, 1/2b and 1/2c was 100%, whereas for serotype 4b it was 98%. Serotyping is a useful tool for first line strain differentiation during epidemiological surveillance and outbreaks. The multiplex PCR assay offers a fast and low-cost alternative, which is easily adaptable to clinical bacteriology and bromatology laboratories.

  9. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    SciTech Connect

    Stulberg, Michael J.; Huang, Qi

    2015-10-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

  10. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether theremore » are also TaqMan/Luminex probe matches within predicted amplicons.« less

  11. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  12. Kneallhazia (=Thelohania) Solenopsae infection rate of Pseudacteon Curvatus flies determined by multiplex PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex PCR method was developed and utilized to determine the Kneallhazia solenopsae infection rate of individual Pseudacteon curvatus flies in north-central Florida. Among P. curvatus flies infected with K. solenopsae, two amplicons were produced, one of 800 nucleotides from the P. curvatus 1...

  13. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    SciTech Connect

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  14. Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye.

    PubMed

    Ahberg, Christian D; Manz, Andreas; Neuzil, Pavel

    2015-01-01

    Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

  15. Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

    PubMed

    Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

    2011-01-01

    In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

  16. Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

    PubMed

    Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

    2011-01-01

    In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize. PMID:22165018

  17. A multiplex reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants.

    PubMed

    Dai, Jin; Cheng, Julong; Huang, Ting; Zheng, Xuan; Wu, Yunfeng

    2012-07-01

    Tobacco viruses including Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tobacco etch virus (TEV), Potato virus Y (PVY) and Tobacco vein banding mosaic virus (TVBMV) are major viruses infecting tobacco and can cause serious crop losses. A multiplex reverse transcription polymerase chain reaction assay was developed to detect simultaneously and differentiate all five viruses. The system used specific primer sets for each virus producing five distinct fragments 237, 273, 347, 456 and 547 bp, representing TMV, CMV subgroup I, TEV, PVY(O) and TVBMV, respectively. These primers were used for detection of the different viruses by single PCR and multiplex PCR and the results were confirmed by DNA sequencing analysis. The protocol was used to detect viruses from different parts of China. The simultaneous and sensitive detection of different viruses using the multiplex PCR is more efficient and economical than other conventional methods for tobacco virus detection. This multiplex PCR provides a rapid and reliable method for the detection and identification of major tobacco viruses, and will be useful for epidemiological studies.

  18. Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

    PubMed Central

    Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

  19. Unyvero i60 implant and tissue infection (ITI) multiplex PCR system in diagnosing periprosthetic joint infection.

    PubMed

    Hischebeth, Gunnar T R; Randau, Thomas M; Buhr, Johanna K; Wimmer, Matthias D; Hoerauf, Achim; Molitor, Ernst; Bekeredjian-Ding, Isabelle; Gravius, Sascha

    2016-02-01

    Periprosthetic joint infection (PJI) is one of the most challenging complications in orthopedic surgery. In cases of suspected periprosthetic joint infection several diagnostic methods are available. In this study we investigated the performance of the newly available Unyvero i60 implant and tissue infection (ITI) multiplex PCR System. 62 specimens from 31 patients with suspected PJI or aseptic loosening of a painful joint arthoplasty were included in this study. Besides the established diagnostic procedures we included a commercial multiplex PCR detection system for diagnosis of PJI. The PCR results obtained from analysis of sonication and synovial fluids (62 specimens) showed a sensitivity of 66.7%, a specificity of 100%, a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 68.4% when compared to cultural methods. Notably, cultures from sonication fluid displayed a sensitivity of 88.9%, a specificity of 61.5%, a PPV of 76.2% and a NPV of 80.0% when compared to tissue cultures. In conclusion, multiplex PCR is an additional - rapid - method for diagnosing PJI. Positive results with the PCR assay used in this study were always confirmed by subsequent matching culture positivity. However, apart from the time saved the nucleic acid amplification technique did not yield additional information than that obtained from microbiological cultures. PMID:26689142

  20. The role of multiplex PCR test in identification of bacterial pathogens in lower respiratory tract infections

    PubMed Central

    Aydemir, Ozlem; Aydemir, Yusuf; Ozdemir, Mehmet

    2014-01-01

    Objectives: Lower respiratory tract infection is one of the most important causes of morbidity and mortality. However establishing a microbial diagnosis for patients with lower respiratory tract infection is still challenging and is often achieved in only half of cases by conventional methods. This study was designed to compare the fast responsive PCR method with the culture method in lower respiratory tract infections and to evaluate the reliability of multiplex PCR method. Methods: One hundred ninety seven patients with the symptoms of acute lower respiratory tract infection, and diagnosed with community-acquired pneumonia, acute exacerbation of chronic obstructive pulmonary disease and exacerbations of bronchiectasis were included in the study. Both culture and PCR methods was performed for the isolation of most commonly seen bacteria, from sputum, nasopharyngeal swabs and bronchoalveolar lavage fluid samples. Results: While at least one bacterial isolation was determined in 62 (31.5%) of all patients with culture method, this number increased to 125 (63.5%) with multiplex PCR. The bacteria most commonly identified by PCR were S. pneumoniae (32%) and H. influenzae (31%). There was a significant difference between PCR and culture in terms of multi-factor detection rates (p<0.005). Multiple bacteria were detected in only two cases in cultures; however, multiple pathogens were detected in 47 cases with PCR. Conclusions: Conventional methods, such as culture and serology are not always adequate to detect the pathogens in lower respiratory tract. Real-time PCR assays proved highly sensitive and rapid. The prevalence of bacteria and multiple agent detected by real-time PCR compared with culture was substantially higher. Widespread use of PCR methods, by providing the immediate and appropriate ''agent specific antibiotic treatment'' of LRTI, will help reduce failure and contributes to a reduction in antibiotic resistance. PMID:25225517

  1. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    PubMed

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences.

  2. Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan

    PubMed Central

    SHIMOJIMA, Yukako; IDA, Miki; NISHINO, Yukari; ISHITSUKA, Rie; KURODA, Sumiyo; HIRAI, Akihiko; SADAMASU, Kenji; NAKAMA, Akiko; KAI, Akemi

    2015-01-01

    PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb. PMID:26537550

  3. 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

  4. Multiplex real-time PCR assay for Legionella species.

    PubMed

    Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

    2015-12-01

    Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies.

  5. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with INNO-LiPA HPV genotyping extra assay.

    PubMed

    Else, Elizabeth A; Swoyer, Ryan; Zhang, Yuhua; Taddeo, Frank J; Bryan, Janine T; Lawson, John; Van Hyfte, Inez; Roberts, Christine C

    2011-05-01

    Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.

  6. A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

    PubMed Central

    2012-01-01

    Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele

  7. A multiplex PCR for the simultaneous detection of Tenacibaculum maritimum and Edwardsiella tarda in aquaculture.

    PubMed

    Castro, Nuria; Toranzo, Alicia E; Magariños, Beatriz

    2014-06-01

    A specific and sensitive multiplex PCR (mPCR) method was developed as a useful tool for the simultaneous detection of two important flatfish pathogens in marine aquaculture, Tenacibaculum maritimum and Edwardsiella tarda. In fish tissues, the average detection limit for these mPCR-amplified organisms was 2 × 10 ⁵ ± 0.2 CFU/g and 4 × 10 ⁵ ± 0.3 CFU/g, respectively. These values are similar or even lower than those previously obtained using the corresponding single PCR. Moreover, mPCR did not produce any nonspecific amplification products when tested against 36 taxonomically related and unrelated strains belonging to 33 different bacterial species. Large amounts of DNA from one of the target bacterial species in the presence of low amounts from the other did not have a significant effect on the amplification sensitivity of the latter.

  8. New Multiplex PCR for Rapid Detection of Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates

    PubMed Central

    Herrera-León, Laura; Molina, Tamara; Saíz, Pilar; Sáez-Nieto, Juan Antonio; Soledad Jiménez, Maria

    2005-01-01

    In this study, we describe a multiplex PCR to detect a AGC→ACC (serine to threonine) mutation in the katG gene and a −15 C-to-T substitution (inhAC−15T) at the 5′ end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur. PMID:15616288

  9. Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections

    PubMed Central

    Chavada, Ruchir; Maley, Michael

    2015-01-01

    Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods. PMID:26464612

  10. Large scale multiplex PCR improves pathogen detection by DNA microarrays

    PubMed Central

    2009-01-01

    Background Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. Results To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000. Conclusion Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR. PMID:19121223

  11. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

    SciTech Connect

    Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

    2000-05-05

    Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

  12. Evaluation of a multiplex real-time PCR method for detecting shiga toxin-producing Escherichia coli in beef and comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology laboratory guidebook method.

    PubMed

    Fratamico, Pina M; Wasilenko, Jamie L; Garman, Bradley; Demarco, Daniel R; Varkey, Stephen; Jensen, Mark; Rhoden, Kyle; Tice, George

    2014-02-01

    The "top-six" non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was ≥1.23 × 10(3) CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted

  13. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

    PubMed Central

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  14. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    PubMed

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

  15. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    PubMed

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya. PMID:26666186

  16. Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

    2005-05-18

    To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

  17. Development of a multiplex PCR-ELISA method for the genetic authentication of Thunnus species and Katsuwonus pelamis in food products.

    PubMed

    Santaclara, Francisco J; Velasco, Amaya; Pérez-Martín, Ricardo I; Quinteiro, Javier; Rey-Méndez, Manuel; Pardo, Miguel Angel; Jimenez, Elisa; Sotelo, Carmen G

    2015-08-01

    In the present work a PCR-ELISA technique for the authentication of Thunnus species was developed. This method is composed by four systems that can be used in a hierarchical way allowing the identification of several scombroids species; or each individual system independently. The hierarchical strategy, proposes a first step, to assign one sample to the Thunnus genus. Next, if the result is positive, several tests can be applied to assign the sample to some particular species of the Thunnus genus. In the case that the result is negative (absence of Thunnus species), it is possible to verify if Katsuwonus pelamis is included in the sample. The method even allows the detection of mixtures of these species in relatively low amounts (up to 1%). Finally, this method was applied to 11 commercial samples to verify the labelling status of tuna products in the market, detecting that 18% were mislabelling.

  18. Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle.

    PubMed

    Junlong, Liu; Li, Youquan; Liu, Aihong; Guan, Guiquan; Xie, Junren; Yin, Hong; Luo, Jianxun

    2015-07-01

    Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

  19. Multiplex PCR To Identify Macrolide Resistance Determinants in Mannheimia haemolytica and Pasteurella multocida

    PubMed Central

    Rose, Simon; Desmolaize, Benoit; Jaju, Puneet; Wilhelm, Cornelia; Warrass, Ralf

    2012-01-01

    The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm(42), which encodes a monomethyltransferase and confers a type I MLSB (macrolide, lincosamide, and streptogramin B) phenotype; msr(E), which encodes a macrolide efflux pump; and mph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm(42), msr(E), and mph(E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA (rrl) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm(42), msr(E), and mph(E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing. PMID:22564832

  20. Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis.

    PubMed

    Lekhak, Sunil Prasad; Sharma, Laxmi; Rajbhandari, Reema; Rajbhandari, Pravesh; Shrestha, Resha; Pant, Basant

    2016-09-01

    Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients. 102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M. tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC. Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%. Multiplex PCR system using primers targeting IS6110 and MPB64, for the detection of M. tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples.

  1. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    SciTech Connect

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  2. Guidelines for Optimisation of a Multiplex Oligonucleotide Ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array

    PubMed Central

    Wuyts, Véronique; Roosens, Nancy H. C.; Marchal, Kathleen; De Keersmaecker, Sigrid C. J.

    2015-01-01

    With multiplex oligonucleotide ligation-PCR (MOL-PCR) different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in high-throughput format. As such, MOL-PCR is a convenient solution for microbial detection and identification assays where many markers should be analysed, including for routine further characterisation of an identified microbial pathogenic isolate. For an assay aimed at routine use, optimisation in terms of differentiation between positive and negative results and of cost and effort is indispensable. As MOL-PCR includes a multiplex ligation step, followed by a singleplex PCR and analysis with microspheres on a Luminex device, several parameters are accessible for optimisation. Although MOL-PCR performance may be influenced by the markers used in the assay and the targeted bacterial species, evaluation of the method of DNA isolation, the probe concentration, the amount of microspheres, and the concentration of reporter dye is advisable in the development of any MOL-PCR assay. Therefore, we here describe our observations made during the optimisation of a 20-plex MOL-PCR assay for subtyping of Salmonella Typhimurium with the aim to provide a possible workflow as guidance for the development and optimisation of a MOL-PCR assay for the characterisation of other microbial pathogens. PMID:25705689

  3. Impact of multiplex PCR on antimicrobial treatment in febrile neutropenia: a randomized controlled study.

    PubMed

    Idelevich, Evgeny A; Silling, Gerda; Niederbracht, Yvonne; Penner, Hanna; Sauerland, Maria Cristina; Tafelski, Sascha; Nachtigall, Irit; Berdel, Wolfgang E; Peters, Georg; Becker, Karsten

    2015-10-01

    Multiplex PCR (mPCR) directly from blood has been suggested as a promising method for rapid identification of pathogens causing sepsis. This study aimed to investigate whether mPCR has any impact on antimicrobial treatment. Hematological patients with febrile neutropenia were randomized into two groups. In the study group, mPCR was performed as an addition to standard diagnostics, and PCR finding was immediately communicated to the clinicians, thus being available for decision making. In the control group, clinicians were not aware of PCR result. PCR samples were collected simultaneously with clinically indicated blood culture specimens from peripheral vein and/or central venous catheter at fever onset and once again if fever persisted up to 72 h. Overall, 74 patients of the study group and 76 patients of the control group were enrolled and 253 samples collected. Therapy was changed to targeted antimicrobial therapy (AMT) in 12 patients (16.2%) in the study group and in 12 patients (15.8%) in the control group. For patients with changes, the median time to change to the targeted AMT was 21.4 h in the study group and 47.5 h in the control group (p = 0.018). In the study group, 57.1% (8/14) of changes to targeted AMT was due to PCR finding. PCR led to AMT change in 9.5% (7/74) of study group patients, i.e., in 33.3% (7/21) of patients who had positive PCR finding. There were no significant differences in patient outcomes (secondary endpoints). In conclusion, PCR method accelerates change to the targeted AMT in febrile neutropenic patients.

  4. Disentangling mite predator-prey relationships by multiplex PCR.

    PubMed

    Pérez-Sayas, Consuelo; Pina, Tatiana; Gómez-Martínez, María A; Camañes, Gemma; Ibáñez-Gual, María V; Jaques, Josep A; Hurtado, Mónica A

    2015-11-01

    Gut content analysis using molecular techniques can help elucidate predator-prey relationships in situations in which other methodologies are not feasible, such as in the case of trophic interactions between minute species such as mites. We designed species-specific primers for a mite community occurring in Spanish citrus orchards comprising two herbivores, the Tetranychidae Tetranychus urticae and Panonychus citri, and six predatory mites belonging to the Phytoseiidae family; these predatory mites are considered to be these herbivores' main biological control agents. These primers were successfully multiplexed in a single PCR to test the range of predators feeding on each of the two prey species. We estimated prey DNA detectability success over time (DS50), which depended on the predator-prey combination and ranged from 0.2 to 18 h. These values were further used to weight prey detection in field samples to disentangle the predatory role played by the most abundant predators (i.e. Euseius stipulatus and Phytoseiulus persimilis). The corrected predation value for E. stipulatus was significantly higher than for P. persimilis. However, because this 1.5-fold difference was less than that observed regarding their sevenfold difference in abundance, we conclude that P. persimilis is the most effective predator in the system; it preyed on tetranychids almost five times more frequently than E. stipulatus did. The present results demonstrate that molecular tools are appropriate to unravel predator-prey interactions in tiny species such as mites, which include important agricultural pests and their predators.

  5. Disentangling mite predator-prey relationships by multiplex PCR.

    PubMed

    Pérez-Sayas, Consuelo; Pina, Tatiana; Gómez-Martínez, María A; Camañes, Gemma; Ibáñez-Gual, María V; Jaques, Josep A; Hurtado, Mónica A

    2015-11-01

    Gut content analysis using molecular techniques can help elucidate predator-prey relationships in situations in which other methodologies are not feasible, such as in the case of trophic interactions between minute species such as mites. We designed species-specific primers for a mite community occurring in Spanish citrus orchards comprising two herbivores, the Tetranychidae Tetranychus urticae and Panonychus citri, and six predatory mites belonging to the Phytoseiidae family; these predatory mites are considered to be these herbivores' main biological control agents. These primers were successfully multiplexed in a single PCR to test the range of predators feeding on each of the two prey species. We estimated prey DNA detectability success over time (DS50), which depended on the predator-prey combination and ranged from 0.2 to 18 h. These values were further used to weight prey detection in field samples to disentangle the predatory role played by the most abundant predators (i.e. Euseius stipulatus and Phytoseiulus persimilis). The corrected predation value for E. stipulatus was significantly higher than for P. persimilis. However, because this 1.5-fold difference was less than that observed regarding their sevenfold difference in abundance, we conclude that P. persimilis is the most effective predator in the system; it preyed on tetranychids almost five times more frequently than E. stipulatus did. The present results demonstrate that molecular tools are appropriate to unravel predator-prey interactions in tiny species such as mites, which include important agricultural pests and their predators. PMID:25824504

  6. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR. PMID:27143320

  7. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

  8. Improved Diagnosis of Periprosthetic Joint Infection by Multiplex PCR of Sonication Fluid from Removed Implants▿

    PubMed Central

    Achermann, Yvonne; Vogt, Markus; Leunig, Michael; Wüst, Jürg; Trampuz, Andrej

    2010-01-01

    The microbiological diagnosis of periprosthetic joint infection (PJI) is crucial for successful antimicrobial treatment. Cultures have limited sensitivity, especially in patients receiving antibiotics. We evaluated the value of multiplex PCR for detection of microbial DNA in sonication fluid from removed orthopedic prostheses. Cases of PJI in which the prosthesis (or part of it) was removed were prospectively included. The removed implant was sonicated, and the resulting sonication fluid was cultured and subjected to multiplex PCR. Of 37 PJI cases (17 hip prostheses, 14 knee prostheses, 4 shoulder prostheses, 1 elbow prosthesis, and 1 ankle prosthesis), pathogens were identified in periprosthetic tissue in 24 (65%) cases, in sonication fluid in 23 (62%) cases, and by multiplex PCR in 29 (78%) cases. The pathogen was detected in 5 cases in sonication fluid only (Propionibacterium acnes in all cases; none of these patients had previously received antibiotics) and in 11 cases by multiplex PCR only (all of these patients had previously received antibiotics). After exclusion of 8 cases caused by P. acnes or Corynebacterium species, which cannot be detected due to the absence of specific primers in the PCR kit, sonication cultures were positive in 17 cases and multiplex PCR sonication cultures were positive in 29 cases (59% versus 100%, respectively; P < 0.01). Among 19 cases (51%) receiving antibiotics, multiplex PCR was positive in all 19 (100%), whereas sonication cultures grew the organism in 8 (42%) (P < 0.01). Multiplex PCR of sonication fluid is a promising test for diagnosis of PJI, particularly in patients who previously received antibiotics. With modified primer sets, multiplex PCR has the potential for further improvement of the diagnosis of PJI. PMID:20164283

  9. Evaluation of a Multiplex Real-time PCR Assay for the Detection of Respiratory Viruses in Clinical Specimens

    PubMed Central

    Rheem, Insoo; Park, Joowon; Kim, Tae-Hyun

    2012-01-01

    Background In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea). Methods Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses. Results The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%. Conclusions The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR. PMID:23130338

  10. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

    PubMed

    Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A

    2016-07-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  11. Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System.

    PubMed

    Rocchetti, Talita T; Silbert, Suzane; Gostnell, Alicia; Kubasek, Carly; Widen, Raymond

    2016-06-01

    A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

  12. High throughput multiplex-PCR for direct detection and diagnosis of dermatophyte species, Candida albicans and Candida parapsilosis in clinical specimen.

    PubMed

    Vahidnia, Ali; Bekers, Wouter; Bliekendaal, Harry; Spaargaren, Joke

    2015-06-01

    We have developed and validated a multiplex-PCR method for detection of dermatophyte spp., Candida albicans and parapsilosis for routine diagnostics. Our m-PCR showed excellent concordance with culture results in 475 clinical samples. Through the rapid diagnosis by our m-PCR, clinicians are able to initiate adequate antimycotic therapy much earlier.

  13. Molecular differentiation of Treponema pallidum subspecies in skin ulceration clinically suspected as yaws in Vanuatu using real-time multiplex PCR and serological methods.

    PubMed

    Chi, Kai-Hua; Danavall, Damien; Taleo, Fasihah; Pillay, Allan; Ye, Tun; Nachamkin, Eli; Kool, Jacob L; Fegan, David; Asiedu, Kingsley; Vestergaard, Lasse S; Ballard, Ronald C; Chen, Cheng-Yen

    2015-01-01

    We developed a TaqMan-based real-time quadriplex polymerase chain reaction (PCR) to simultaneously detect Treponema pallidum subspecies pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum, the causative agents of venereal syphilis, yaws, and bejel, respectively. The PCR assay was applied to samples from skin ulcerations of clinically presumptive yaws cases among children on Tanna Island, Vanuatu. Another real-time triplex PCR was used to screen for the point mutations in the 23S rRNA genes that have previously been associated with azithromycin resistance in T. pallidum subsp. pallidum strains. Seropositivity by the classical syphilis serological tests was 35.5% among children with skin ulcerations clinically suspected with yaws, whereas the presence of T. pallidum subsp. pertenue DNA was only found in lesions from 15.5% of children. No evidence of T. pallidum subsp. pertenue infection, by either PCR or serology was found in ∼59% of cases indicating alternative causes of yaws-like lesions in this endemic area.

  14. Universal primer-multiplex PCR approach for simultaneous detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in food samples.

    PubMed

    Yuan, Yanfang; Xu, Wentao; Zhai, Zhifang; Shi, Hui; Luo, Yunbo; Chen, Zhuojun; Huang, Kunlun

    2009-10-01

    Escherichia coli, Listeria monocytogenes, and Salmonella spp. are 3 kinds of the most important food-borne human pathogens. Traditional microbiological analysis is labor-intensive, time-consuming, and easily contaminated, thus producing false positive signals; it also involves much subjectivity judgments. Multiplex-PCR could be applied to detect multiple target organisms simultaneously to save time and labor, but there is always disproportionate amplification resulting from the disparity of different primers. To gain a rapid and sensitive method, a universal primer-multiplex PCR system (UP-M-PCR) was developed and applied for simultaneous detection of the 3 organisms. This method simplified traditional multiplex-PCR reaction system and overcame its amplification disparities among different primers; moreover, it got a high specificity and sensitivity (85, 155, and 104 copies/reaction for E. coli O157, L. monocytogenes, and Salmonella spp., respectively). Compared with the time-consuming and laborious microbiological analysis, UP-M-PCR had a lower risk of cross-contamination without inoculation and incubation. Test results for 36 food samples showed that UP-M-PCR method got a relative accuracy of 91.77% when compared with traditional microbiological analysis. It could serve as a rapid screening method for pathogen detection and could detect target genes even in dead pathogenic cells. In addition, it has the potential to be performed in an automation mode and might find broader application in simultaneous detection of other multiple pathogens.

  15. Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex.

    PubMed

    Spositto, F L E; Campanerut, P A Z; Ghiraldi, L D; Leite, C Q F; Hirata, M H; Hirata, R D C; Siqueira, V L D; Cardoso, R Fressatti

    2014-01-01

    We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

  16. A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera).

    PubMed

    Hamiduzzaman, Mollah Md; Guzman-Novoa, Ernesto; Goodwin, Paul H

    2010-10-01

    Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R(2)=0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.

  17. Advances in multiplex PCR: balancing primer efficiencies and improving detection success

    PubMed Central

    Sint, Daniela; Raso, Lorna; Traugott, Michael

    2012-01-01

    1. Multiplex PCR is a valuable tool in many biological studies but it is a multifaceted procedure that has to be planned and optimised thoroughly to achieve robust and meaningful results. In particular, primer concentrations have to be adjusted to assure an even amplification of all targeted DNA fragments. Until now, total DNA extracts were used for balancing primer efficiencies; however, the applicability for comparisons between taxa or different multiple-copy genes was limited owing to the unknown number of template molecules present per total DNA. 2. Based on a multiplex system developed to track trophic interactions in high Alpine arthropods, we demonstrate a fast and easy way of generating standardised DNA templates. These were then used to balance the amplification success for the different targets and to subsequently determine the sensitivity of each primer pair in the multiplex PCR. 3. In the current multiplex assay, this approach led to an even amplification success for all seven targeted DNA fragments. Using this balanced multiplex PCR, methodological bias owing to variation in primer efficiency will be avoided when analysing field-derived samples. 4. The approach outlined here allows comparing multiplex PCR sensitivity, independent of the investigated species, genome size or the targeted genes. The application of standardised DNA templates not only makes it possible to optimise primer efficiency within a given multiplex PCR, but it also offers to adjust and/or to compare the sensitivity between different assays. Along with other factors that influence the success of multiplex reactions, and which we discuss here in relation to the presented detection system, the adoption of this approach will allow for direct comparison of multiplex PCR data between systems and studies, enhancing the utility of this assay type. PMID:23549328

  18. Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Jia; Miao, Haizhen; Wu, Houfei; Huang, Wensheng; Tang, Rong; Qiu, Minyan; Wen, Jianguo; Zhu, Shuifang; Li, Yao

    2006-07-15

    In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95% of currently commercial GMO plants and the limits of detection are 0.5% for soybean and 1% for maize. This method is proved to be a new method for routine analysis of GMOs.

  19. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    PubMed Central

    Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

    2016-01-01

    In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

  20. An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails.

    PubMed

    Caron, Y; Righi, S; Lempereur, L; Saegerman, C; Losson, B

    2011-05-31

    This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react. PMID:21242033

  1. An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails.

    PubMed

    Caron, Y; Righi, S; Lempereur, L; Saegerman, C; Losson, B

    2011-05-31

    This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react.

  2. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters.

    PubMed

    Wadle, Simon; Lehnert, Michael; Schuler, Friedrich; Köppel, René; Serr, Annerose; Zengerle, Roland; von Stetten, Felix

    2016-01-01

    Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.

  3. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters.

    PubMed

    Wadle, Simon; Lehnert, Michael; Schuler, Friedrich; Köppel, René; Serr, Annerose; Zengerle, Roland; von Stetten, Felix

    2016-01-01

    Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength. PMID:27625206

  4. Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay.

    PubMed

    Zhao, H X; Li, Z J; Hu, S W; Sun, G L; Chang, J J; Zhang, Z H

    2010-08-01

    Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed. PMID:20401459

  5. Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR.

    PubMed

    Schrell, Adrian M; Roper, Michael G

    2014-06-01

    A frequency-modulated fluorescence encoding method was used as a means to increase the number of fluorophores monitored during infrared-mediated polymerase chain reaction. Laser lines at 488 nm and 561 nm were modulated at 73 and 137 Hz, respectively, exciting fluorescence from the dsDNA intercalating dye, EvaGreen, and the temperature insensitive dye, ROX. Emission was collected in a color-blind manner using a single photomultiplier tube for detection and demodulated by frequency analysis. The resulting frequency domain signal resolved the contribution from the two fluorophores as well as the background from the IR lamp. The detection method was successfully used to measure amplification of DNA samples containing 10(4)-10(7) starting copies of template producing an amplification efficiency of 96%. The utility of this methodology was further demonstrated by simultaneous amplification of two genes from human genomic DNA using different color TaqMan probes. This method of multiplexing fluorescence detection with IR-qPCR is ideally suited as it allows isolation of the signals of interest from the background in the frequency domain and is expected to further reduce the complexity of multiplexed microfluidic IR-qPCR instrumentation.

  6. Development of a multiplexed PCR detection method for Barley and Cereal yellow dwarf viruses, Wheat spindle streak virus, Wheat streak mosaic virus and Soil-borne wheat mosaic virus.

    PubMed

    Deb, Mahua; Anderson, Joseph M

    2008-03-01

    Barley and Cereal yellow dwarf viruses (B/CYDVs), Wheat spindle streak mosaic (WSSMV), Soil-borne wheat mosaic virus (SBWMV) and Wheat streak mosaic virus (WSMV) constitute the most economically important group of wheat viruses. In this paper, a multiplex reverse transcription polymerase chain reaction (M-RT-PCR) method was developed for the simultaneous detection and discrimination of eight viruses: five strains of B/CYDVs, WSSMV, SBWMV and WSMV. The protocol uses specific primer sets for each virus producing five distinct fragments 295, 175, 400, 237, and 365 bp, indicating the presence of two strains of BYDVs, -PAV, -MAV, CYDV-RPV and two unassigned Luteoviridae BYDV-SGV and -RMV, respectively. This system also readily detected WSSMV, SBWMV and WSMV specific amplicons at 154, 219 and 193 bp, respectively. The amplification specificity of these primers was tested against a range of field samples from different parts of United States. The protocol also utilizes fluorescently tagged primers that can streamline the detection of each virus through capillary electrophoresis. This study fulfills the need for a rapid and specific wheat virus diagnostic tool that also has the potential for investigating the epidemiology of these viral diseases.

  7. Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment.

    PubMed

    Pélandakis, Michel; Pernin, Pierre

    2002-04-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.

  8. A multiplex PCR protocol for rapid identification of Candida glabrata and its phylogenetically related species Candida nivariensis and Candida bracarensis.

    PubMed

    Romeo, Orazio; Scordino, Fabio; Pernice, Ida; Lo Passo, Carla; Criseo, Giuseppe

    2009-10-01

    We have developed a multiplex PCR protocol for the detection of Candida glabrata and its closely related species Candida nivariensis and Candida bracarensis. The method uses four PCR primers, targeting the ITS1 region and the 5.8S ribosomal RNA gene. The combination of these primers yielded unique results to all Candida species tested. The PCR assay we developed was found to be a rapid, specific and easy to perform method and it will be useful for characterizing large numbers of isolates for epidemiological studies.

  9. 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

  10. Internal transcribed spacer guided multiplex PCR for species identification of Convolvulus prostratus and Evolvulus alsinoides.

    PubMed

    Sharma, Sonal; Shrivastava, Neeta

    2016-05-01

    Shankhpushpi is a reputed drug from an Indian system of medicine for treating mental disorders and enhancing memory. Two herbs, namely Convolvulus prostratus Forssk. and Evolvulus alsinoides (L.) L., are commonly known as Shankhpushpi. Ambiguous vernacular identity can affect the scientific validity of the Shankpushpi-based herbal drug therapy. In the present investigation, a novel and sensitive multiplex PCR method based on polymorphism in the internal transcribed spacer (ITS) region was developed to establish the molecular identity of C. prostratus and E. alsinoides. DNA was isolated and the ITS region was amplified, sequenced and assembled. Sequences were aligned to identify variable nucleotides in order to develop plant-specific primers. Primers were validated in singleplex reactions and eventually a multiplex assay was developed. This assay was tested for sensitivity and validated by amplifying DNA isolated from the simulated blended powdered plant material. Primers developed for C. prostratus resulted into a 200 bp amplicon and 596 bp for E. alsinoides. The assay was found to be sensitive enough for amplification of low quantities of DNA. The method can detect 10% of the mixing of plants with each other in blended material. This PCR assay can be used for rapid botanical identification of Shankhpushpi plant materials and will improve evidence-based herbal drug therapy. PMID:27175337

  11. Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.

    PubMed

    Anniballi, Fabrizio; Auricchio, Bruna; Delibato, Elisabetta; Antonacci, Monia; De Medici, Dario; Fenicia, Lucia

    2012-01-27

    Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.

  12. Internal transcribed spacer guided multiplex PCR for species identification of Convolvulus prostratus and Evolvulus alsinoides

    PubMed Central

    Sharma, Sonal; Shrivastava, Neeta

    2016-01-01

    Shankhpushpi is a reputed drug from an Indian system of medicine for treating mental disorders and enhancing memory. Two herbs, namely Convolvulus prostratus Forssk. and Evolvulus alsinoides (L.) L., are commonly known as Shankhpushpi. Ambiguous vernacular identity can affect the scientific validity of the Shankpushpi-based herbal drug therapy. In the present investigation, a novel and sensitive multiplex PCR method based on polymorphism in the internal transcribed spacer (ITS) region was developed to establish the molecular identity of C. prostratus and E. alsinoides. DNA was isolated and the ITS region was amplified, sequenced and assembled. Sequences were aligned to identify variable nucleotides in order to develop plant-specific primers. Primers were validated in singleplex reactions and eventually a multiplex assay was developed. This assay was tested for sensitivity and validated by amplifying DNA isolated from the simulated blended powdered plant material. Primers developed for C. prostratus resulted into a 200 bp amplicon and 596 bp for E. alsinoides. The assay was found to be sensitive enough for amplification of low quantities of DNA. The method can detect 10% of the mixing of plants with each other in blended material. This PCR assay can be used for rapid botanical identification of Shankhpushpi plant materials and will improve evidence-based herbal drug therapy. PMID:27175337

  13. A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

    PubMed

    Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

    2015-07-01

    Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing. PMID:26016439

  14. A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

    PubMed

    Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

    2015-07-01

    Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing.

  15. Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing.

    PubMed

    Chen, Zhiyao; Liu, Yunlong; Duan, Wenbang; Ye, Hui; Wu, Haiping; Li, Jinheng; Zhou, Guohua

    2014-06-01

    Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.

  16. Molecular diagnostics of the honey bee parasites Lotmaria passim and Crithidia spp. (Trypanosomatidae) using multiplex PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lotmaria passim Schwarz is a recently described trypanosome parasite of honey bees in continental United States, Europe, and Japan. We developed a multiplex PCR technique using a PCR primer specific for L. passim to distinguish this species from C. mellificae. We report the presence of L. passim in ...

  17. Molecular-beacon multiplex real-time PCR assay for detection of Vibrio cholerae.

    PubMed

    Gubala, Aneta J; Proll, David F

    2006-09-01

    A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

  18. Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

    PubMed Central

    Tsouma, Aikaterini; Aggeli, Chrysanthi; Lembessis, Panagiotis; Zografos, George N; Korkolis, Dimitris P; Pectasides, Dimitrios; Skondra, Maria; Pissimissis, Nikolaos; Tzonou, Anastasia; Koutsilieris, Michael

    2010-01-01

    AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting circulating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors’ clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P < 0.001) and preoperative serum levels of CEA (P = 0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented significant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RT-PCR assay can provide useful information concerning disease stage and overall survival of CRC patients. PMID:21157973

  19. Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA.

    PubMed

    Zebardast, Nozhat; Yeganeh, Farshid; Gharavi, Mohammad Javad; Abadi, Alireza; Seyyed Tabaei, Seyyed Javad; Haghighi, Ali

    2016-10-01

    Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study

  20. Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA.

    PubMed

    Zebardast, Nozhat; Yeganeh, Farshid; Gharavi, Mohammad Javad; Abadi, Alireza; Seyyed Tabaei, Seyyed Javad; Haghighi, Ali

    2016-10-01

    Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study

  1. Development and validation of a multiplex reverse transcription PCR assay for simultaneous detection of three papaya viruses.

    PubMed

    Tuo, Decai; Shen, Wentao; Yang, Yong; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-10-21

    Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay's specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

  2. Development of a multiplex PCR for identification of Dictyocaulus lungworms in domestic and wild ruminants.

    PubMed

    Pyziel, Anna M; Laskowski, Zdzisław; Höglund, Johan

    2015-10-01

    Dictyocaulus lungworms are the causative agents of parasitic bronchitis (dictyocaulosis) characterised by coughing and severe lung pathology in domestic and wild ruminants. The objective of this study was to design a simple molecular test that could detect of lungworm DNA from both adult and larval lungworms and could distinguish between the most common Dictyocaulus species found in cattle and in some species of wild ruminants. A multiplex PCR test with four novel primers targeting species-specific regions of the second internal transcribed spacer (ITS2) was designed based on our own sequence data as well as on available sequence information in GenBank. After PCR amplification of lungworms from European bison (Bison bonasus), cattle (Bos taurus), moose (Alces alces), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), products were analysed with gel electrophoresis. This resulted in three specific bands of different size depending on the species analysed. Dictyocaulus viviparus collected from cattle or European bison resulted in a ca. 560 bp band, D. capreolus collected from roe deer produced a band ca. 400 bp and the longest DNA band (ca. 660 bp) was obtained with DNA from Dictyocaulus sp. collected from red deer and moose. Dictyocaulus eckerti bands with expected size of 714 bp were not observed in our study. The multiplex method produced consistent results with samples from both Sweden and Poland and overcame the limitations of traditional techniques based on differences in morphological features of parasites at different life stages. PMID:26266883

  3. Detection of four important Eimeria species by multiplex PCR in a single assay.

    PubMed

    You, Myung-Jo

    2014-06-01

    The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl.

  4. Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers.

    PubMed

    Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C; Chorianopoulos, Nikos

    2015-10-22

    Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

  5. Development of a multiplex PCR for identification of Dictyocaulus lungworms in domestic and wild ruminants.

    PubMed

    Pyziel, Anna M; Laskowski, Zdzisław; Höglund, Johan

    2015-10-01

    Dictyocaulus lungworms are the causative agents of parasitic bronchitis (dictyocaulosis) characterised by coughing and severe lung pathology in domestic and wild ruminants. The objective of this study was to design a simple molecular test that could detect of lungworm DNA from both adult and larval lungworms and could distinguish between the most common Dictyocaulus species found in cattle and in some species of wild ruminants. A multiplex PCR test with four novel primers targeting species-specific regions of the second internal transcribed spacer (ITS2) was designed based on our own sequence data as well as on available sequence information in GenBank. After PCR amplification of lungworms from European bison (Bison bonasus), cattle (Bos taurus), moose (Alces alces), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), products were analysed with gel electrophoresis. This resulted in three specific bands of different size depending on the species analysed. Dictyocaulus viviparus collected from cattle or European bison resulted in a ca. 560 bp band, D. capreolus collected from roe deer produced a band ca. 400 bp and the longest DNA band (ca. 660 bp) was obtained with DNA from Dictyocaulus sp. collected from red deer and moose. Dictyocaulus eckerti bands with expected size of 714 bp were not observed in our study. The multiplex method produced consistent results with samples from both Sweden and Poland and overcame the limitations of traditional techniques based on differences in morphological features of parasites at different life stages.

  6. Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

    PubMed Central

    Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

    2016-01-01

    The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process. PMID:27239109

  7. A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones.

    PubMed

    Martins, Natacha; Picão, Renata Cristina; Cerqueira-Alves, Morgana; Uehara, Aline; Barbosa, Lívia Carvalho; Riley, Lee W; Moreira, Beatriz Meurer

    2016-08-01

    A collection of 163 Acinetobacter baumannii isolates detected in a large Brazilian hospital, was potentially related with the dissemination of four clonal complexes (CC): 113/79, 103/15, 109/1 and 110/25, defined by University of Oxford/Institut Pasteur multilocus sequence typing (MLST) schemes. The urge of a simple multiplex-PCR scheme to specify these clones has motivated the present study. The established trilocus sequence-based typing (3LST, for ompA, csuE and blaOXA-51-like genes) multiplex-PCR rapidly identifies international clones I (CC109/1), II (CC118/2) and III (CC187/3). Thus, the system detects only one (CC109/1) out of four main CC in Brazil. We aimed to develop an alternative multiplex-PCR scheme to detect these clones, known to be present additionally in Africa, Asia, Europe, USA and South America. MLST, performed in the present study to complement typing our whole collection of isolates, confirmed that all isolates belonged to the same four CC detected previously. When typed by 3LST-based multiplex-PCR, only 12% of the 163 isolates were classified into groups. By comparative sequence analysis of ompA, csuE and blaOXA-51-like genes, a set of eight primers was designed for an alternative multiplex-PCR to distinguish the five CC 113/79, 103/15, 109/1, 110/25 and 118/2. Study isolates and one CC118/2 isolate were blind-tested with the new alternative PCR scheme; all were correctly clustered in groups of the corresponding CC. The new multiplex-PCR, with the advantage of fitting in a single reaction, detects five leading A. baumannii clones and could help preventing the spread in healthcare settings.

  8. Xenomonitoring of different filarial nematodes using single and multiplex PCR in mosquitoes from Assiut Governorate, Egypt.

    PubMed

    Dyab, Ahmed Kamal; Galal, Lamia Ahmed; Mahmoud, Abeer El-Sayed; Mokhtar, Yasser

    2015-02-01

    Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs. PMID:25748712

  9. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

    PubMed Central

    Te, Shu Harn; Chen, Enid Yingru

    2015-01-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  10. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

    PubMed

    Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

    2015-08-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.

  11. [Application of the molecular test PCR multiplex for identification of Mycobacterium bovis BCG strains].

    PubMed

    Augustynowicz-Kopeć, Ewa; Zabost, Anna; Brzezińiska, Sylwia; Wasowicz, Marcin; Zwolska, Zofia

    2005-01-01

    In our last paper (18) we described the problem of proper microbiological identification of BCG strains and how important is distinguishing vaccine strain from virulent strains of Mycobacterium tuberculosis complex. We have suggested the modern algorithm of BCG strains identification including mycolic acids profile by HPLC and 14C PZA resistance methods. These methods allowed us to made fast and accurate microbiological identification of side effects of BCG vaccine in the children. Identification of BCG by HPLC is possible within one working day compared with 3-4 weeks required for conventional methods. However both methods need very expensive instruments like HPLC and/or Bactec-460 Tb radiometric system. Presently we have evaluated molecular test based on the analyzis of the region RD1 encoding a 9.5-kb fragment. This fragment is deleted in all BCG substrains (6) and present in all human and bovine virulent strains. To evaluate this method for the rapid and specific detection of BCG, a large strain collection (32 strains) representating M. bovis BCG (vaccine strains and strains isolated from the children in case of adverse reactions after vaccination) M. bovis and M. tuberculosis from own collection was analyzed. RD1 was present in all 15 M. tuberculosis and M. bovis tested strains and deleted in 17 of 18 BCG strains. The multiplex PCR method was 100% sensitive and specific for the identification of BCG among strains of the Mycobacterium tuberculosis complex. Multiplex PCR can be used as a diagnostic test and has significant advantages over existing methods. PMID:16989158

  12. Detection of free-living amoebae by using multiplex quantitative PCR.

    PubMed

    Le Calvez, Thomas; Trouilhé, Marie-Cécile; Humeau, Philippe; Moletta-Denat, Marina; Frère, Jacques; Héchard, Yann

    2012-06-01

    Free-living amoebae (FLA) are protozoa found worldwide in soil and aquatic environments, which are able to colonize man-made water networks. Some FLA have the potential to be pathogenic and others might harbour pathogenic bacteria. Indeed, FLA feed on bacteria, but some bacteria could resist phagocytosis and either survive in FLA or even multiply within FLA. These bacteria are collectively named amoeba resistant bacteria (ARB). The best characterized example is Legionella pneumophila, for which FLA is the main reservoir in the environment. Not only could FLA be a reservoir that protects ARB, some bacteria might become more resistant to treatment and be more virulent. Thus, it is of medical significance to quantify FLA populations in soil, water or the environment. The main limitation for the quantification of FLA is that classical culture is not efficient and reliable for many genera and 'strains'. Thus, several PCR-based quantification methods have been published for various FLA. However, thus far, no method has been published to simultaneously quantify the main FLA genera in the same PCR reaction. In this study, we developed a multiplex qPCR method to detect both Amoebozoan (i.e. Acanthamoeba, Hartmannella and Echinamoeba) and Vahlkampfiidae (i.e. Vahlkampfia and Naegleria) using 18S ribosomal RNA as the target gene. This method was shown to be specific, reliable and sensitive, could be used for the quantification of FLA and is likely to be useful to anticipate risks due to FLA or pathogenic bacteria, such as L. pneumophila.

  13. Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt.

    PubMed

    Nazeer, John T; El Sayed Khalifa, Khalifa; von Thien, Heidrun; El-Sibaei, Mahmoud Mohamed; Abdel-Hamid, Magda Youssef; Tawfik, Ranya Ayman Samir; Tannich, Egbert

    2013-02-01

    Diarrhea is an important cause of morbidity and mortality, worldwide. Giardia intestinalis, Cryptosporidium spp., and Entamoeba histolytica are the most common diarrhea-causing parasitic protozoa. Diagnosis of these parasites is usually performed by microscopy. However, microscopy lacks sensitivity and specificity. Replacing microscopy with more sensitive and specific nucleic acid based methods is hampered by the higher costs, in particular in developing countries. Multiplexing the detection of more than one parasite in a single test by real-time polymerase chain reaction (PCR) has been found to be very effective and would decrease the cost of the test. In the present study, stool samples collected from 396 Egyptian patients complaining of diarrhea along with 202 faecal samples from healthy controls were examined microscopically by direct smear method and after concentration using formol-ethyl acetate. Frozen portions of the same samples were tested by multiplex real-time for simultaneous detection of E. histolytica, G. intestinalis, and Cryptosporidium spp. The results indicate that among diarrheal patients in Egypt G. intestinalis is the most common protozoan parasite, with prevalence rates of 30.5 and 37.1 %, depending on the method used (microscopy vs. multiplex real-time PCR). Cryptosporidium spp. was detected in 1 % of the diarrheal patients by microscopy and in 3 % by real-time PCR. While E. histolytica/dispar was detected in 10.8 % by microscopy, less than one fifth of them (2 %) were found true positive for Entamoeba dispar by real-time PCR. E. histolytica DNA was not detected in any of the diarrheal patients. In comparison with multiplex real-time PCR, microscopy exhibited many false positive and negative cases with the three parasites giving sensitivities and specificities of 100 and 91 % for E. histolytica/dispar, 57.8 and 85.5 % for G. intestinalis, and 33.3 and 100 % for Cryptosporidium spp.

  14. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

    PubMed Central

    Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

    2016-01-01

    Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the

  15. A high-throughput multiplex method adapted for GMO detection.

    PubMed

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  16. Simultaneous typing of seven porcine pathogens by multiplex PCR with a GeXP analyser.

    PubMed

    Hu, Ling; Lin, Xingyu; Nie, Fuping; ZexiaoYang; Yao, Xueping; Li, Guili; Wu, Xulong; Ren, Meishen; Wang, Yin

    2016-06-01

    A novel high-throughput method was developed for simultaneous detection and differentiation of seven porcine pathogens by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyser. The pathogens included in this study were pseudorabies virus (PRV), classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2) and Japanese encephalitis virus (JEV). Seven pairs of chimeric primers, consisting of a pathogen-specific sequence fused to a universal sequence at the 5' end, were used to initiate the PCR, after which a set of universal primers was used for the subsequent cycles of the PCR. Amplification products were separated by capillary electrophoresis and identified using fluorescence spectrophotometry. The specificity of the GeXP assay was examined with single and mixed pathogen cDNA/DNA templates. The specific DNA product amplification peaks of seven pathogens were observed on the GeXP analyser. Negative controls did not produce DNA products. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of the plasmids containing the target sequence. Under optimised conditions this assay achieved a sensitivity of 100-1000 copies/μL for a single virus and 1000 copies/μL when all of the seven pre-mixed viral targets were present. Furthermore, the GeXP-PCR assay was 100% specific when 58 clinical samples were tested in comparison with the conventional PCR method. In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for simultaneously detecting seven pathogens that infect swine. PMID:26706731

  17. Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen.

    PubMed Central

    Pooler, M R; Ritchie, D F; Hartung, J S

    1996-01-01

    Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria. PMID:8795198

  18. Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants.

    PubMed

    Zhang, Xiao; Liu, Zhijie; Yang, Jifei; Chen, Ze; Guan, Guiquan; Ren, Qiaoyun; Liu, Aihong; Luo, Jianxun; Yin, Hong; Li, Youquan

    2014-02-01

    Infections with Theileria sp. may cause significant economic losses to the sheep industry. Species identification based on microscopic examination is difficult, and more suitable methods are required for the rapid detection and identification of Theileria sp, in clinical specimens. In this study, a multiplex polymerase chain reaction (mPCR) assay was developed to simultaneously identify three individual Theileria species in small ruminants. Three pairs of specific, sensitive primers were designed on the basis of the 5.8S ribosomal RNA gene (Theileria luwenshuni and Theileria ovis) and the 18S ribosomal RNA gene (Theileria uilenbergi) to generate target products of 303, 884, and 530 bp, respectively. Standard DNA for each of the three species was extracted from blood recovered from infected sheep, and a preliminary study was conducted on 56 sheep to verify the reliability of the system. Optimal PCR conditions, including primer concentration, annealing time, and the number of amplification cycles, were established. The assay sensitivity under these conditions was 10(-3) % parasitemia, and its specificity was 100 %. The results of the study suggest that mPCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of Theileria species in small ruminants.

  19. Rapid detection by multiplex PCR of Genomic Islands, prophages and Integrative Conjugative Elements in V. cholerae 7th pandemic variants.

    PubMed

    Spagnoletti, Matteo; Ceccarelli, Daniela; Colombo, Mauro M

    2012-01-01

    Vibrio cholerae poses a threat to human health, and new epidemic variants have been reported so far. Seventh pandemic V. cholerae strains are characterized by highly related genomic sequences but can be discriminated by a large set of Genomic Islands, phages and Integrative Conjugative Elements. Classical serotyping and biotyping methods do not easily discriminate among new variants arising worldwide, therefore the establishment of new methods for their identification is required. We developed a multiplex PCR assay for the rapid detection of the major 7th pandemic variants of V. cholerae O1 and O139. Three specific genomic islands (GI-12, GI-14 and GI-15), two phages (Kappa and TLC), Vibrio Seventh Pandemic Island 2 (VSP-II), and the ICEs of the SXT/R391 family were selected as targets of our multiplex PCR based on a comparative genomic approach. The optimization and specificity of the multiplex PCR was assessed on 5 V. cholerae 7th pandemic reference strains, and other 34 V. cholerae strains from various epidemic events were analyzed to validate the reliability of our method. This assay had sufficient specificity to identify twelve different V. cholerae genetic profiles, and therefore has the potential to be used as a rapid screening method.

  20. Single Multiplex PCR Assay To Identify Simultaneously the Six Categories of Diarrheagenic Escherichia coli Associated with Enteric Infections

    PubMed Central

    Vidal, Maricel; Kruger, Eileen; Durán, Claudia; Lagos, Rosanna; Levine, Myron; Prado, Valeria; Toro, Cecilia; Vidal, Roberto

    2005-01-01

    We designed a multiplex PCR for the detection of all categories of diarrheagenic Escherichia coli. This method proved to be specific and rapid in detecting virulence genes from Shiga toxin-producing (stx1, stx2, and eae), enteropathogenic (eae and bfp), enterotoxigenic (stII and lt), enteroinvasive (virF and ipaH), enteroaggregative (aafII), and diffuse adherent (daaE) Escherichia coli in stool samples. PMID:16208019

  1. Comparison of a multiplex reverse transcription-PCR-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens.

    PubMed

    Liolios, L; Jenney, A; Spelman, D; Kotsimbos, T; Catton, M; Wesselingh, S

    2001-08-01

    A multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.

  2. Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping

    PubMed Central

    Robberts, Lourens; Wolter, Nicole; Nicol, Paul; Mafofo, Joseph; Africa, Samantha; Zar, Heather J.; Nicol, Mark P.

    2015-01-01

    Background Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. Methods A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. Results Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently

  3. Rapid Identification of Mycobacterium Species with the Aid of Multiplex Polymerase Chain Reaction (PCR) From Clinical Isolates

    PubMed Central

    Gupta, Siddhartha; Bandyopadhyay, Debasis; Paine, Suman Kalyan; Gupta, Soma; Banerjee, Surajita; Bhattacharya, Sujata; Gachhui, Ratan; Bhattacharya, Basudev

    2010-01-01

    Mycobacteria are aerobic, nonspore forming, non-motile,single-cell bacteria.Of more than 40 currently recognized species of mycobacteria, Mycobacterium tuberculosis, the causative agent of human TB is the commonest pathogen for pulmonary and extra pulmonary tuberculosis cases. The other members of the Mycobacterium tuberculosis complex (MTC) or the nontubercular mycobacterium (NTM) produces similar diseases which cannot be differentiated from tuberculosis by clinical symptoms and signs. But this differentiation is important as the chemotherapy varies widely according to the strain of mycobacterium. The burden of morbidity and mortality of tuberculosis is rapidly growing worldwide, particularly with the HIV/AIDS epidemic. The strain identification of Mycobacterium remains a cumbersome, labor intensive and expensive procedure, which requires 3 to 12 weeks of time. The conventional methods of strain identification lack proper standardization and precise diagnosis. The prime objective of this study is to overcome these problems. A multiplex PCR using 3 amplicons of 165,365, and 541 base pair target sequences was done with a total number of 165 clinical isolates of suspected Koch’s patients. Strain identification was compared both by conventional methods and multiplex PCR. The results of the study show that this multiplex PCR is supposed to be less complicated, less time consuming, cost-effective and superior to the conventional methods. It is also applicable for culture negative samples where strain identification is not possible by conventional approach. PMID:21258579

  4. Development of a Single-Reaction Multiplex PCR Toxin Typing Assay for Staphylococcus aureus Strains

    PubMed Central

    Sharma, Naresh K.; Rees, Catherine E. D.; Dodd, Christine E. R.

    2000-01-01

    We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes. PMID:10742210

  5. Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes

    PubMed Central

    Barnard, Emma; Nagy, István; Hunyadkürti, Judit; Patrick, Sheila

    2015-01-01

    The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterized by MLST and representing types IA1 (n = 145), IA2 (n = 20), IB (n = 65), IC (n = 7), II (n = 45), and III (n = 30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses. PMID:25631794

  6. [Clarification of a break-in theft crime by multiplex PCR analysis of cigarette butts].

    PubMed

    Hochmeister, M; Haberl, J; Borer, V; Rudin, O; Dirnhofer, R

    1995-01-01

    This paper describes the first use of multiplex PCR amplification kits for the analysis of DNA extracted from cigarette butts in a criminal case. Two suspects could be excluded as potential contributors to the samples, whereas the multi locus PCR-based DNa profile derived from the cigarette butts was consistent with a DNA profile derived from a third suspect. For identity testing in criminal cases where cigarette butts are involved, commercially available PCR amplification kits provide currently the most powerful tool. Furthermore this PCR-based analysis can be implemented into most application orientated laboratories.

  7. Updated Campylobacter jejuni Capsule PCR Multiplex Typing System and Its Application to Clinical Isolates from South and Southeast Asia

    PubMed Central

    Poly, Frédéric; Serichantalergs, Oralak; Kuroiwa, Janelle; Pootong, Piyarat; Mason, Carl; Guerry, Patricia; Parker, Craig T.

    2015-01-01

    Campylobacter jejuni produces a polysaccharide capsule that is the major determinant of the Penner serotyping scheme. This passive slide agglutination typing system was developed in the early 1980’s and was recognized for over two decades as the gold standard for C. jejuni typing. A preliminary multiplex PCR technique covering 17 serotypes was previously developed in order to replace this classic serotyping scheme. Here we report the completion of the multiplex PCR technology that is able to identify all the 47 Penner serotypes types known for C. jejuni. The number of capsule types represented within the 47 serotypes is 35. We have applied this method to a collection of 996 clinical isolates from Thailand, Cambodia and Nepal and were able to successfully determine capsule types of 98% of these. PMID:26630669

  8. Multiplex real-time PCR for rapid Staphylococcal cassette chromosome mec typing.

    PubMed

    Chen, Liang; Mediavilla, José R; Oliveira, Duarte C; Willey, Barbara M; de Lencastre, Herminia; Kreiswirth, Barry N

    2009-11-01

    Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the DeltamecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the DeltamecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.

  9. Development of a multiplex PCR assay to detect Edwardsiella tarda, Streptococcus parauberis, and Streptococcus iniae in olive flounder (Paralichthys olivaceus).

    PubMed

    Park, Seong Bin; Kwon, Kyoung; Cha, In Seok; Jang, Ho Bin; Nho, Seong Won; Fagutao, Fernand F; Kim, Young Kyu; Yu, Jong Earn; Jung, Tae Sung

    2014-01-01

    A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.

  10. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

    PubMed Central

    Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva

    2016-01-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

  11. Multiplex PCR assay for the detection of enterotoxic Bacillus cereus group strains and its application in food matrices.

    PubMed

    Kalyan Kumar, T D; Murali, H S; Batra, H V

    2010-06-01

    Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10(1)-10(2) organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.

  12. Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp.

    PubMed

    Herrera-León, Silvia; McQuiston, John R; Usera, Miguel A; Fields, Patricia I; Garaizar, Javier; Echeita, M Aurora

    2004-06-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z(10), H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, GA, have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz(10), H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously. This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.

  13. Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay.

    PubMed

    Kim, Suk Ran; Ki, Chang-Seok; Lee, Nam Yong

    2009-03-01

    Acute viral respiratory infections are among the most common causes of human disease. Rapid and accurate diagnosis of viral respiratory infections is important for providing timely therapeutic interventions. This study evaluated a new multiplex PCR assay (Seegene Inc., Seoul, Korea) for simultaneous detection and identification of 12 respiratory viruses using two primer mixes. The viruses included parainfluenza viruses 1, 2, and 3, human metapneumovirus, human coronavirus 229E/NL63 and OC43, adenovirus, influenza viruses A and B, human respiratory syncytial viruses A and B, and human rhinovirus A. The analytical sensitivity of the assay was 10-100 copies per reaction for each type of virus. There was no cross-reactivity with common bacterial or viral pathogens. A comparison with conventional viral culture and immunofluorescence was carried out using 101 respiratory specimens from 92 patients. Using viral culture, 57 specimens (56.4%) were positive without co-infection. The same viruses were identified in all 57 specimens using the multiplex PCR. Seven of the 57 specimens (12.3%) were found to be co-infected with other respiratory viruses, and 19 of 44 (43.2%) specimens which were negative by culture were positive by the multiplex PCR. The Seeplex Respiratory Virus Detection assay represents a significant improvement over the conventional methods for the detection of a broad spectrum of respiratory viruses.

  14. NTMG (N-terminal Truncated Mutants Generator for cDNA): an automatic multiplex PCR assays design for generating various N-terminal truncated cDNA mutants.

    PubMed

    Chen, Yung-Fu; Chen, Rung-Ching; Tseng, Lin-Yu; Lin, Elong; Chan, Yung-Kuan; Pan, Ren-Hao

    2007-07-01

    The sequential deletion method is generally used to locate the functional domain of a protein. With this method, in order to find the various N-terminal truncated mutants, researchers have to investigate the ATG-like codons, to design various multiplex polymerase chain reaction (PCR) forward primers and to do several PCR experiments. This web server (N-terminal Truncated Mutants Generator for cDNA) will automatically generate groups of forward PCR primers and the corresponding reverse PCR primers that can be used in a single batch of a multiplex PCR experiment to extract the various N-terminal truncated mutants. This saves much time and money for those who use the sequential deletion method in their research. This server is available at http://oblab.cs.nchu.edu.tw:8080/WebSDL/. PMID:17488836

  15. Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

    SciTech Connect

    Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R.; Miles, R.R.; Brown, S.B.; Stratton, P.L.; Milanovich, F.P.

    2001-04-20

    The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.

  16. Multiplex PCR and RFLP approaches for identification of rabbitfish (Siganus) species using mitochondrial gene regions.

    PubMed

    Ravago-Gotanco, R G; Manglicmot, M T; Pante, M J R

    2010-07-01

    Molecular assays are described for the identification of six rabbitfish (Siganus) species. A multiplex PCR assay using primers targeting the mitochondrial cytochrome b region simultaneously identifies four species: Siganus canaliculatus, S. fuscescens, S. javus, and S. spinus. Subsequent RFLP assays of multiplex amplicons differentiate between S. virgatus and S. corallinus based on diagnostic fragments from the mitochondrial cytochrome oxidase I region. Assays were validated with known specimens demonstrating accuracy of the molecular identification. Applied to morphologically indistinguishable early developmental stages, these assays can facilitate studies on species-specific spatio-temporal patterns of larval dispersal and population connectivity to aid fishery management.

  17. A Novel Multiplex Real-Time PCR for the Identification of Mycobacteria Associated with Zoonotic Tuberculosis

    PubMed Central

    Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Niemann, Stefan; van Soolingen, Dick; Barry, Thomas

    2011-01-01

    Background Tuberculosis (TB) is the leading cause of death worldwide from a single infectious agent. An ability to detect the Mycobacterium tuberculosis complex (MTC) in clinical material while simultaneously differentiating its members is considered important. This allows for the gathering of epidemiological information pertaining to the prevalence, transmission and geographical distribution of the MTC, including those MTC members associated with zoonotic TB infection in humans. Also differentiating between members of the MTC provides the clinician with inherent MTC specific drug susceptibility profiles to guide appropriate chemotherapy. Methodology/Principal Findings The aim of this study was to develop a multiplex real-time PCR assay using novel molecular targets to identify and differentiate between the phylogenetically closely related M. bovis, M. bovis BCG and M. caprae. The lpqT gene was explored for the collective identification of M. bovis, M. bovis BCG and M. caprae, the lepA gene was targeted for the specific identification of M. caprae and a Region of Difference 1 (RD1) assay was incorporated in the test to differentiate M. bovis BCG. The multiplex real-time PCR assay was evaluated on 133 bacterial strains and was determined to be 100% specific for the members of the MTC targeted. Conclusions/Significance The multiplex real-time PCR assay developed in this study is the first assay described for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. Future validation of this multiplex assay should demonstrate its potential in the rapid and accurate diagnosis of TB caused by these three mycobacteria. Furthermore, the developed assay may be used in conjunction with a recently described multiplex real-time PCR assay for identification of the MTC and simultaneous differentiation of M. tuberculosis, M. canettii resulting in an ability to differentiate five of the eight members of the

  18. Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

    PubMed

    Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

    2016-01-01

    Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

  19. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR.

    PubMed

    Gimenes, Fabrícia; Medina, Fabiana Soares; Abreu, André Luelsdorf Pimenta de; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) -1 and -2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks.

  20. Detection and typing of human papillomavirus by e6 nested multiplex PCR.

    PubMed

    Sotlar, K; Diemer, D; Dethleffs, A; Hack, Y; Stubner, A; Vollmer, N; Menton, S; Menton, M; Dietz, K; Wallwiener, D; Kandolf, R; Bültmann, B

    2004-07-01

    A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.

  1. A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

    PubMed Central

    Ogawa, Hirohito; Fujikura, Daisuke; Ohnuma, Miyuki; Ohnishi, Naomi; Hang'ombe, Bernard M.; Mimuro, Hitomi; Ezaki, Takayuki; Mweene, Aaron S.; Higashi, Hideaki

    2015-01-01

    Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis. PMID:25774512

  2. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

    PubMed

    Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

    2004-12-01

    This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

  3. Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

    PubMed Central

    Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

    2004-01-01

    This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

  4. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  5. Detection of intrinsic blaOXA-51-like by multiplex PCR on its own is not reliable for the identification of Acinetobacter baumannii.

    PubMed

    Zander, Esther; Higgins, Paul G; Fernández-González, Ana; Seifert, Harald

    2013-03-01

    Three clinical A. baumannii isolates Ab-508, Ab-511, and Ab-653 were recovered from South Africa, South Korea, and Turkey, respectively. Multiplex PCR to detect OXA-type carbapenemases showed atypical blaOXA-51-like amplification products. The aim of this study was to investigate the background of changes in blaOXA-51-like PCR products. Isolates were confirmed as A. baumannii using gyrB multiplex and rpoB sequencing and were epidemiologically unrelated by rep-PCR-based DiversiLab. Sequencing of blaOXA-51-like revealed an insertion of ISAba15 in blaOXA-66 (isolate Ab-511) and an insertion of the novel ISAba19 in blaOXA-78 (isolates Ab-508 and Ab-653). Detection of the intrinsic blaOXA-51-like by OXA-multiplex PCR should not be considered a fully reliable method for identification of A. baumannii when used without an additional independent method. Other species identification methods such as gyrB multiplex PCR and rpoB sequencing should be used to reliably identify A. baumannii.

  6. Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

    PubMed Central

    Jung, Seungwon; Kim, Junsun; Lee, Dong Jin; Oh, Eun Hae; Lim, Hwasup; Kim, Kwang Pyo; Choi, Nakwon; Kim, Tae Song; Kim, Sang Kyung

    2016-01-01

    Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs). PMID:26964639

  7. Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis.

    PubMed

    James, Delano; Schmidt, Anna-Mary; Wall, Erika; Green, Margaret; Masri, Saad

    2003-09-24

    Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.

  8. Multiplex real-time PCR for identification of canine parvovirus antigenic types.

    PubMed

    Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Narang, Deepti

    2016-07-01

    Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types.

  9. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

    PubMed

    Majumder, S; Baranwal, V K

    2014-06-01

    Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. PMID:24598229

  10. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

    PubMed

    Majumder, S; Baranwal, V K

    2014-06-01

    Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material.

  11. Multiplex-PCR for Identification of Two Species in Genus Hishimonus (Hemiptera: Cicadellidae) in Jujube Orchards.

    PubMed

    Hao, Shaodong; Wang, He; Tao, Wanqiang; Wang, Jinzhong; Zhang, Zhiyong; Zhang, Qiuling; Zhang, Minzhao; Guo, Li; Shi, Xiaoyu

    2015-10-01

    The insect family Cicadellidae includes economically important vectors of plant pathogens. Hishimonus sellatus (Uhler) transmits jujube witches'-broom (JWB). Currently, H. sellatus and Hishimonus lamellatus Cai et Kuoh are observed to co-occur at the same locality on jujube. H. lamellatus is now suspected to be a JWB vector. As such, correct identification of Hishimonus species present in vineyards is essential for epidemiological surveys. However, traditional identification of Hishimonus by morphology is limited to the adult male. We provide a comprehensive description of morphological and molecular tools for discriminating between H. sellatus and H. lamellatus, for use in identification and monitoring of the two Hishimonus species and studies of their plant hosts. A rapid and inexpensive method is introduced to identify H. sellatus and H. lamellatus occurring in jujube orchards. This method is based on amplification of mitochondrial cytochrome oxidase I (COI) gene, using PCR with multiplexed, species-specific primers. The reliability of this new method has been tested on different populations from different sites in Beijing region of China. PMID:26453733

  12. Multiplex PCR To Diagnose Bloodstream Infections in Patients Admitted from the Emergency Department with Sepsis ▿

    PubMed Central

    Tsalik, Ephraim L.; Jones, Daphne; Nicholson, Bradly; Waring, Lynette; Liesenfeld, Oliver; Park, Lawrence P.; Glickman, Seth W.; Caram, Lauren B.; Langley, Raymond J.; van Velkinburgh, Jennifer C.; Cairns, Charles B.; Rivers, Emanuel P.; Otero, Ronny M.; Kingsmore, Stephen F.; Lalani, Tahaniyat; Fowler, Vance G.; Woods, Christopher W.

    2010-01-01

    Sepsis is caused by a heterogeneous group of infectious etiologies. Early diagnosis and the provision of appropriate antimicrobial therapy correlate with positive clinical outcomes. Current microbiological techniques are limited in their diagnostic capacities and timeliness. Multiplex PCR has the potential to rapidly identify bloodstream infections and fill this diagnostic gap. We identified patients from two large academic hospital emergency departments with suspected sepsis. The results of a multiplex PCR that could detect 25 bacterial and fungal pathogens were compared to those of blood culture. The results were analyzed with respect to the likelihood of infection, sepsis severity, the site of infection, and the effect of prior antibiotic therapy. We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis, and 14% had septic shock. The majority had a definite infection (41.5%) or a probable infection (30.7%). Blood culture and PCR performed similarly with samples from patients with clinically defined infections (areas under the receiver operating characteristic curves, 0.64 and 0.60, respectively). However, blood culture identified more cases of septicemia than PCR among patients with an identified infectious etiology (66 and 46, respectively; P = 0.0004). The two tests performed similarly when the results were stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who had previously received antibiotics (P = 0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification. PMID:19846634

  13. Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another prim...

  14. Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay

    PubMed Central

    Li, Li; Yan, Hong-Bin; Blair, David; Lei, Meng-Tong; Cai, Jin-Zhong; Fan, Yan-Lei; Li, Jian-Qiu; Fu, Bao-Quan; Yang, Yu-Rong; McManus, Donald P.; Jia, Wan-Zhong

    2015-01-01

    Background Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification. Methodology/Principal Findings A single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively. Conclusions/Significance The multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification

  15. A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis.

    PubMed

    Alba, Annia; Vázquez, Antonio A; Hernández, Hilda; Sánchez, Jorge; Marcet, Ricardo; Figueredo, Mabel; Sarracent, Jorge; Fraga, Jorge

    2015-07-30

    Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis - F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere. PMID:26012858

  16. A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis.

    PubMed

    Alba, Annia; Vázquez, Antonio A; Hernández, Hilda; Sánchez, Jorge; Marcet, Ricardo; Figueredo, Mabel; Sarracent, Jorge; Fraga, Jorge

    2015-07-30

    Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis - F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere.

  17. Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR.

    PubMed

    Maas, L; Dorigo-Zetsma, J W; de Groot, C J; Bouter, S; Plötz, F B; van Ewijk, B E

    2014-06-01

    The performance of a multiplex real-time PCR for the detection of Blastocystis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium species and Entamoeba species in faecal samples was evaluated in an observational prospective study. Paediatric patients (0-18 years) presenting with gastrointestinal symptoms and suspected of having enteroparasitic disease were included. A questionnaire on gastrointestinal symptoms and the chosen treatment was completed at the start of the study and after 6 weeks. Of 163 paediatric patients (mean age, 7.8 years), 114 (70%) had a PCR-positive faecal sample. D. fragilis was detected most frequently, in 101 patients, followed by Blastocystis in 49. In faecal samples of 47 patients, more than one protozoan was detected, mainly the combination of D. fragilis and Blastocystis. Reported gastrointestinal symptoms were abdominal pain (78%), nausea (30%), and altered bowel habits (28%). Eighty-nine of the PCR-positive patients were treated with antibiotics. A significant reduction in abdominal pain was observed both in treated and in untreated patients. This study demonstrated that multiplex real-time PCR detects a high percentage of intestinal protozoa in paediatric patients with gastrointestinal symptoms. However, interpretation and determination of the clinical relevance of a positive PCR result in this population are still difficult.

  18. Identification of high-risk Listeria monocytogenes serotypes in lineage I (serotype 1/2a, 1/2c, 3a and 3c) using multiplex PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: Using molecular subtyping techniques, Listeria monocytogenes is divided into three major phylogenetic lineages, and a multiplex PCR method can differentiate five L. monocytogenes subgroups: 1/2a-3a, 1/2c-3c, 1/2b-3b-7, 4b-4d-4e, and 4a-4c. In the current study, we conducted genome comparison...

  19. Microbiological Diagnosis of Severe Diarrhea in Kidney Transplant Recipients by Use of Multiplex PCR Assays

    PubMed Central

    Coste, Jean-François; Vuiblet, Vincent; Moustapha, Betoul; Bouin, Alexis; Lavaud, Sylvie; Toupance, Olivier; de Rougemont, Alexis; Benejat, Lucie; Megraud, Francis; Wolak-Thierry, Aurore; Villena, Isabelle; Chemla, Cathy; Le Magrex, Elisabeth; de Champs, Christophe; Andreoletti, Laurent; Rieu, Philippe

    2013-01-01

    Diarrhea is a frequent complication after kidney transplantation, ascribed to adverse effects of the immunosuppressive therapy in case of negative microbiological examination of the stools. The aim of this study was to improve the microbiological diagnosis by implementing molecular tests. Fifty-four severe diarrhea events that occurred in 49 adult kidney transplant recipients from September 2010 to November 2011 were investigated. One or several enteric pathogens were detected in 13 (23%) stool samples using classical microbiological methods versus 39 (72%) for the seven commercially available multiplex PCR assays used retrospectively (P = 0.006). Interestingly, molecular diagnosis identified 15 multiple infections compared to none using classical techniques. The primary pathogens detected were enteropathogenic Escherichia coli (EPEC) (n = 15; 38%), Campylobacter spp. (n = 15; 38%), and Norovirus (n = 14; 36%). Specificities for Campylobacter and Norovirus infection diagnosis were 75 and 100%, respectively, by comparison to reference methods. Based on molecular findings, a cyclosporine-mycophenolate mofetil combination was identified as a risk factor for developing Norovirus-induced diarrhea. Norovirus infections were also responsible for higher weight loss than all the other causes of diarrhea. In samples from asymptomatic immunocompromised and immunocompetent patients, EPEC but not Norovirus and Campylobacter infections were detected at a frequency similar to that observed in symptomatic kidney transplant recipients. In conclusion, molecular tools significantly improved the detection of single and multiple enteric infections by comparison to classical techniques and could quickly become the key element in the management of severe acute diarrhea in transplant recipients. PMID:23554205

  20. Multiplex PCR for Distinguishing the Most Common Phase-1 Flagellar Antigens of Salmonella spp.

    PubMed Central

    Herrera-León, Silvia; McQuiston, John R.; Usera, Miguel A.; Fields, Patricia I.; Garaizar, Javier; Echeita, M. Aurora

    2004-01-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z10, H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, Ga., have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz10, H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously (P. G. Agron, R. L. Walker, H. Kinde, S. J. Sawyer, D. C. Hayes, J. Wollard, and G. L. Andersen, Appl. Environ. Microbiol. 67:4984-4991, 2001). This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested. PMID:15184437

  1. Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

    PubMed Central

    Peters, Sarah E.; Wang, Jinhong; Hernandez-Garcia, Juan; Weinert, Lucy A.; Luan, Shi-Lu; Chaudhuri, Roy R.; Angen, Øystein; Aragon, Virginia; Williamson, Susanna M.; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Maskell, Duncan J.; Tucker, Alexander W.

    2015-01-01

    Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis. PMID:26424843

  2. Multiplex PCR Assay for Detection of Vibrio vulnificus Biotype 2 and Simultaneous Discrimination of Serovar E Strains▿

    PubMed Central

    Sanjuán, Eva; Amaro, Carmen

    2007-01-01

    In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples. PMID:17277209

  3. A Simple Multiplex PCR Approach for Target Enrichment in Next-Gen Sequencing

    PubMed Central

    Zhu, Qi; Hebel, Chris; Zhou, Xiaochun

    2014-01-01

    Multiplexing PCR is a simple way to extract genomic regions of interest for various medical and genetic tests. Somatic mutations lead to various diseases including cancer. These mutations are unlikely to be best detected using regular whole genome sequencing. Clinical samples often consist of disease cells, e.g. cancer cells, surrounded by normal cells. Thus, deep sequencing of hundreds to thousands fold coverage is required to detect the mutations. In clinical research many doctors are interested in specific genes or genomic regions and they want to extract the regions from genomic DNA or RNA before sequencing. Many current clinical, forensic, and heretical genetic test workflows start with multiplexing PCR to extract genetic marker carrying regions from whole genomes before running hybridization, sequencing, or electrophoresis tests to identify the markers. Personal medicine and prognosis mostly involve examining sequence variations of a number of targeted genes and metabolic pathway genes so as to predict drug efficacies and drug toxicities. We have developed a new multiplexing PCR approach with a significantly simplified workflow and significantly improved robustness. When applied to sequencing target enrichment application, the workflow for producing amplified targets involves only one hands-on step and one PCR run. The approach is designed to require low sample input and to produce superior amplicon uniformity and sequence specificity. The approach involves a novel primer design and a proprietary reaction composition. A PCR run consists of two functionally separated reaction phases, namely target capture and library amplification, without any hands-on step in between. The performance of the new approach will be demonstrated by a caner panel data.

  4. Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses.

    PubMed

    Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

    2016-10-01

    Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/μL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups in pigs. PMID:27448821

  5. Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control.

    PubMed

    Wei, Shuang; Zhao, Hui; Xian, Yuyin; Hussain, Malik A; Wu, Xiyang

    2014-06-01

    A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10(5) CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.

  6. Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

    PubMed

    Ranjan, R K; Singh, Dinesh; Baranwal, V K

    2016-11-01

    Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale. PMID:27480266

  7. Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

    PubMed

    Ranjan, R K; Singh, Dinesh; Baranwal, V K

    2016-11-01

    Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

  8. A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples.

    PubMed

    Lee, Hwan Young; Yoon, Jung Ah; Yang, Woo Ick; Shin, Kyoung-Jin

    2013-01-01

    The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups.

  9. Multiplex coherent raman spectroscopy detector and method

    NASA Technical Reports Server (NTRS)

    Chen, Peter (Inventor); Joyner, Candace C. (Inventor); Patrick, Sheena T. (Inventor); Guyer, Dean R. (Inventor)

    2004-01-01

    A multiplex coherent Raman spectrometer (10) and spectroscopy method rapidly detects and identifies individual components of a chemical mixture separated by a separation technique, such as gas chromatography. The spectrometer (10) and method accurately identify a variety of compounds because they produce the entire gas phase vibrational Raman spectrum of the unknown gas. This is accomplished by tilting a Raman cell (20) to produce a high-intensity, backward-stimulated, coherent Raman beam of 683 nm, which drives a degenerate optical parametric oscillator (28) to produce a broadband beam of 1100-1700 nm covering a range of more than 3000 wavenumber. This broadband beam is combined with a narrowband beam of 532 nm having a bandwidth of 0.003 wavenumbers and focused into a heated windowless cell (38) that receives gases separated by a gas chromatograph (40). The Raman radiation scattered from these gases is filtered and sent to a monochromator (50) with multichannel detection.

  10. Multiplex coherent raman spectroscopy detector and method

    DOEpatents

    Chen, Peter; Joyner, Candace C.; Patrick, Sheena T.; Guyer, Dean R.

    2004-06-08

    A multiplex coherent Raman spectrometer (10) and spectroscopy method rapidly detects and identifies individual components of a chemical mixture separated by a separation technique, such as gas chromatography. The spectrometer (10) and method accurately identify a variety of compounds because they produce the entire gas phase vibrational Raman spectrum of the unknown gas. This is accomplished by tilting a Raman cell (20) to produce a high-intensity, backward-stimulated, coherent Raman beam of 683 nm, which drives a degenerate optical parametric oscillator (28) to produce a broadband beam of 1100-1700 nm covering a range of more than 3000 wavenumber. This broadband beam is combined with a narrowband beam of 532 nm having a bandwidth of 0.003 wavenumbers and focused into a heated windowless cell (38) that receives gases separated by a gas chromatograph (40). The Raman radiation scattered from these gases is filtered and sent to a monochromator (50) with multichannel detection.

  11. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  12. Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis

    PubMed Central

    Raj, Ankush; Singh, Netrapal; Gupta, Krishna B.; Chaudhary, Dhruva; Yadav, Aparna; Chaudhary, Anil; Agarwal, Kshitij; Varma-Basil, Mandira; Prasad, Rajendra; Khuller, Gopal K.

    2016-01-01

    Purpose Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. Materials and Methods We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. Results Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. Conclusion These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease. PMID:26632387

  13. A multiplex-PCR assay for identification of the quarantine plant pathogen Xanthomonas axonopodis pv. phaseoli.

    PubMed

    Boureau, T; Kerkoud, M; Chhel, F; Hunault, G; Darrasse, A; Brin, C; Durand, K; Hajri, A; Poussier, S; Manceau, C; Lardeux, F; Saubion, F; Jacques, M-A

    2013-01-01

    In this study we developed an algorithm to screen for all exact molecular signatures of the quarantine pathogen Xanthomonas axonopodis pv. phaseoli (Xap), based on available data of the presence or absence of virulence-associated genes. The simultaneous presence of genes avrBsT and xopL is specific to Xap. Therefore we developed a multiplex PCR assay targeting avrBsT and xopL for the molecular identification of Xap. The specificity of this multiplex was validated by comparison to that of other molecular identification assays aimed at Xap, on a wide collection of reference strains. This multiplex was further validated on a blind collection of Xanthomonas isolates for which pathogenicity was assayed by stem wounding and by dipping leaves into calibrated inocula. This multiplex was combined to the previously described X4c/X4e molecular identification assay for Xap. Such a combination enables the molecular identification of all strains of Xanthomonas pathogenic on bean. Results also show that assay by stem wounding does not give reliable results in the case of Xap, and that pathogenicity assays by dipping should be preferred. PMID:23142341

  14. Modified midi- and mini-multiplex PCR systems for mitochondrial DNA control region sequence analysis in degraded samples.

    PubMed

    Kim, Na Young; Lee, Hwan Young; Park, Sun Joo; Yang, Woo Ick; Shin, Kyoung-Jin

    2013-05-01

    Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60-year-old and 400-500-year-old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.

  15. A Multiplex PCR-coupled Liquid Bead Array for the Simultaneous Detection of Four Biothreat Agents

    SciTech Connect

    Wilson, W J; Erler, A M; Nasarabadi, S L; Skowronski, E W; McCready, P M

    2004-02-04

    We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species -specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high- throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3,000 individual data points within a single 8-hour shift for approximately $1.20 per sample in a 10-plexed assay.

  16. Rapid identification of Histoplasma capsulatum directly from cultures by multiplex PCR.

    PubMed

    Elías, Nahuel Alejandro; Cuestas, María Luján; Sandoval, Macarena; Poblete, Gabriela; Lopez-Daneri, Gabriela; Jewtuchowicz, Virginia; Iovannitti, Cristina; Mujica, María Teresa

    2012-12-01

    The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I-Hc II) and a region of approximately 600-bp (ITS1-ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100 % sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I-Hc II in the presence of the ITS1-ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100 %. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis.

  17. Rapid identification of Histoplasma capsulatum directly from cultures by multiplex PCR.

    PubMed

    Elías, Nahuel Alejandro; Cuestas, María Luján; Sandoval, Macarena; Poblete, Gabriela; Lopez-Daneri, Gabriela; Jewtuchowicz, Virginia; Iovannitti, Cristina; Mujica, María Teresa

    2012-12-01

    The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I-Hc II) and a region of approximately 600-bp (ITS1-ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100 % sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I-Hc II in the presence of the ITS1-ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100 %. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis. PMID:22821346

  18. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays.

    PubMed

    Call, D R; Brockman, F J; Chandler, D P

    2001-07-20

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml(-1) (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass-based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products; the system is amenable to automation.

  19. Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J. ); Chandler, Darrell P.

    2000-12-01

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  20. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

    2001-07-05

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  1. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

    PubMed

    Fleming, Rachel I; Harbison, SallyAnn

    2010-07-01

    With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain.

  2. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

    PubMed

    Fleming, Rachel I; Harbison, SallyAnn

    2010-07-01

    With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain. PMID:20457026

  3. Multiplex PCR for Differential Identification of Broad Tapeworms (Cestoda: Diphyllobothrium) Infecting Humans▿

    PubMed Central

    Wicht, Barbara; Yanagida, Tetsuya; Scholz, Tomáš; Ito, Akira; Jiménez, Juan A.; Brabec, Jan

    2010-01-01

    The specific identification of broad tapeworms (genus Diphyllobothrium) infecting humans is very difficult to perform by morphological observation. Molecular analysis by PCR and sequencing represents the only reliable tool to date to identify these parasites to the species level. Due to the recent spread of human diphyllobothriosis in several countries, a correct diagnosis has become crucial to better understand the distribution and the life cycle of human-infecting species as well as to prevent the introduction of parasites to disease-free water systems. Nevertheless, PCR and sequencing, although highly precise, are too complicated, long, and expensive to be employed in medical laboratories for routine diagnostics. In the present study we optimized a cheap and rapid molecular test for the differential identification of the most common Diphyllobothrium species infecting humans (D. latum, D. dendriticum, D. nihonkaiense, and D. pacificum), based on a multiplex PCR with the cytochrome c oxidase subunit 1 gene of mitochondrial DNA. PMID:20592146

  4. Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

    PubMed

    Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

    2012-11-01

    Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

  5. A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

    PubMed Central

    Fraga, Aline Padilha; de Vargas, Tatiana; Ikuta, Nilo; Fonseca, André Salvador Kazantzi; Celmer, Álvaro José; Marques, Edmundo Kanan; Lunge, Vagner Ricardo

    2013-01-01

    Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories. PMID:24294247

  6. Pathotyping of Vibrio isolates by multiplex PCR reveals a risk of virulent strain spreading in New Caledonian shrimp farms.

    PubMed

    Labreuche, Yannick; Pallandre, Laurane; Ansquer, Dominique; Herlin, José; Wapotro, Billy; Le Roux, Frédérique

    2012-01-01

    Two recurring syndromes threaten the viability of the shrimp industry in New Caledonia, which represents the second largest export business. The "Syndrome 93" is a cold season disease due to Vibrio penaeicida affecting all shrimp farms, while the "Summer Syndrome" is a geographically restricted vibriosis caused by a virulent lineage of Vibrio nigripulchritudo. Microbiological procedures for diagnosis of these diseases are time-consuming and do not have the ability to discriminate the range of virulence potentials of V. nigripulchritudo. In this study, we developed a multiplex PCR method to simultaneously detect these two bacterial species and allow for pathotype discrimination. The detection limits of this assay, that includes an internal amplification control to eliminate any false-negative results, were determined at 10 pg purified DNA and 200 cfu/ml. After confirming the effectiveness of our method using experimentally infected animals, its accuracy was compared to standard biochemical methods during a field survey using 94 samples collected over 3 years from shrimp farms encountering mortality events. The multiplex PCR showed very high specificity for the detection of V. penaeicida and V. nigripulchritudo (inclusivity and exclusivity 100%) and allowed us to detect the spreading of highly pathogenic isolates of V. nigripulchritudo to a farm adjoining the "Summer Syndrome area." This assay represents a simple, rapid, and cost-effective diagnostic tool for implementing timely risk management decisions but also understanding the seasonal and geographical distribution of these pathogens. PMID:22001997

  7. The effect of multiplex-PCR-assessed major pathogens causing subclinical mastitis on somatic cell profiles.

    PubMed

    Goli, Mohammad; Ezzatpanah, Hamid; Ghavami, Mehrdad; Chamani, Mohammad; Aminafshar, Mehdi; Toghiani, Majid; Eghbalsaied, Shahin

    2012-10-01

    The major pathogens causing mastitis were evaluated by multiplex-polymerase chain reaction (M-PCR) with self-designed primers in four quarters of the first, third, and fifth parities in industrial, semi-industrial, and traditional dairy cattle farms in Iran. With the incidence of infection in the quarters by Staphylococcus aureus and Streptococcus agalactiae, the mean log somatic cell count (log SCC) increased from 5.06 to 5.77. The smallest changes occurred with Escherichia coli. Contagious pathogens, when compared with environmental pathogens, were more prevalent and common and created more profound quantitative and qualitative changes in SCC profiles. The second part of the study surveyed the diversity of contaminating pathogens and their effect on quantitative and qualitative profiles of somatic cells. M-PCR was used to determine the absence (M-PCR(-)) and presence of one (M-PCR(+1)), two (M-PCR(+2)), and three (M-PCR(+3)) major pathogens in raw milk samples. Quarter log SCC increased from 5.06 (for M-PCR(-1)) to 5.5 (for M-PCR(+1)), 5.7 (for M-PCR(+2)), and 6 (for M-PCR(+3)). Percent changes in polymorphonuclears (PMNs) were not significant between different quarters and parities but were significant between different farms in terms of pathogen diversity (P < 0.05). Therefore, by increasing the number of types of major pathogens involved in subclinical mastitis, SCC of udder quarters and the proportion of PMNs significantly increased, whereas the proportion of lymphocytes significantly decreased. This subject is very important in increasing the shelf life of dairy products, because PMNs are introduced to the enzymatic pools. PMID:22535149

  8. Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

    PubMed

    Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

    2003-09-01

    A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains. PMID:12958274

  9. A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae).

    PubMed

    Saccaggi, D L; Krüger, K; Pietersen, G

    2008-02-01

    Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.

  10. Simultaneous detection and differentiation of three viruses in pear plants by a multiplex RT-PCR.

    PubMed

    Yao, Bingyu; Wang, Guoping; Ma, Xiaofang; Liu, Wenbin; Tang, Huihui; Zhu, Hui; Hong, Ni

    2014-02-01

    A multiplex RT-PCR (mRT-PCR) assay was developed for detection and differentiation of the Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV), which are viruses frequently occurring in pear trees. Different combinations of mixed primer pairs were tested for their specificity and sensitivity for the simultaneous detection of the three viruses. Three primer pairs were used to amplify their fragments of 247bp, 358bp and 500bp, respectively. The primer pair for ASPV was designed in this work, while the primer pairs for ACLSV and ASGV were from previous reports. The sensitivity and specificity of the mRT-PCR assay for the three viruses were comparable to that of each uniplex RT-PCR. The mRT-PCR was applied successfully for the detection of three viruses in leaves of pear and apple plants, but was unreliable in the detection of ASGV in dormant barks. In conclusion, this mRT-PCR provides a useful tool for the routine and rapid detection and the differentiation of three pear viruses.

  11. Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India.

    PubMed

    Sumbria, Deepak; Singla, L D; Sharma, Amrita; Bal, M S; Kumar, Sanjay

    2015-07-30

    Multiplex PCR for simultaneous detection of Trypanosoma evansi and Theileria equi in single-step reaction was optimized and employed on 108 equids (99 horses and 9 donkeys/mules) blood samples collected from two agro-climatic zones (Sub-mountain undulating zone and Undulating plain zone) of Punjab to evaluate the status of concurrent infection and associated risk factors. The amplification products of 257 and 709 bp targeting repetitive nucleotide sequence of variable surface glycoproteins of T. evansi and 18S rRNA gene of T. equi, respectively expressed high fidelity of the primer pairs with sequence homology to neighboring geographic isolates. The overall prevalence of T. evansi and T. equi was 3.7 and 1.85%, with Undulating plain zone at higher infection risk for T. equi (OR=3.24, 95% CI=0.28-83.65); and Sub-mountain undulating zone (OR=∞, 95% CI=0.25-∞) for T. evansi. Multiplex PCR revealed higher risk of infection of both T. equi (OR=6.75, 95% CI=0.58-175.38) and T. evansi (OR=2.11, 95% CI=0.05-80.36) in the farms with inappropriate management system. The risk factor associated with the type of host species had an odds ratio of 12.35 (95% CI=0.29-508.37) for donkeys/mules versus horses for T. evansi infection. This group was also at higher risk of infection with Odds ratio (OR) of 4 (95% CI=0.14-53.99) for T. equi. The current investigation brings out various commodities at risk of infection pertaining to equid trypanosomosis and theileriosis evaluated by a rapid and sensitive multiplex PCR assay.

  12. Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

  13. Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene, cgcA, To Differentiate Species within the Genus Cronobacter

    PubMed Central

    Carter, L.; Lindsey, L. A.; Grim, C. J.; Sathyamoorthy, V.; Jarvis, K. G.; Gopinath, G.; Lee, C.; Sadowski, J. A.; Trach, L.; Pava-Ripoll, M.; McCardell, B. A.; Tall, B. D.

    2013-01-01

    In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations. PMID:23144142

  14. Developmental validation of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit: an established multiplex assay with improved performance.

    PubMed

    Wang, Dennis Y; Chang, Chien-Wei; Lagacé, Robert E; Calandro, Lisa M; Hennessy, Lori K

    2012-03-01

    Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit is an improved version of the AmpFℓSTR(®) Identifiler(®) PCR Amplification Kit and amplifies the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit for human identity and parentage testing.

  15. Delay grid multiplexing: simple time-based multiplexing and readout method for silicon photomultipliers

    NASA Astrophysics Data System (ADS)

    Won, Jun Yeon; Ko, Guen Bae; Lee, Jae Sung

    2016-10-01

    In this paper, we propose a fully time-based multiplexing and readout method that uses the principle of the global positioning system. Time-based multiplexing allows simplifying the multiplexing circuits where the only innate traces that connect the signal pins of the silicon photomultiplier (SiPM) channels to the readout channels are used as the multiplexing circuit. Every SiPM channel is connected to the delay grid that consists of the traces on a printed circuit board, and the inherent transit times from each SiPM channel to the readout channels encode the position information uniquely. Thus, the position of each SiPM can be identified using the time difference of arrival (TDOA) measurements. The proposed multiplexing can also allow simplification of the readout circuit using the time-to-digital converter (TDC) implemented in a field-programmable gate array (FPGA), where the time-over-threshold (ToT) is used to extract the energy information after multiplexing. In order to verify the proposed multiplexing method, we built a positron emission tomography (PET) detector that consisted of an array of 4  ×  4 LGSO crystals, each with a dimension of 3  ×  3  ×  20 mm3, and one- to-one coupled SiPM channels. We first employed the waveform sampler as an initial study, and then replaced the waveform sampler with an FPGA-TDC to further simplify the readout circuits. The 16 crystals were clearly resolved using only the time information obtained from the four readout channels. The coincidence resolving times (CRTs) were 382 and 406 ps FWHM when using the waveform sampler and the FPGA-TDC, respectively. The proposed simple multiplexing and readout methods can be useful for time-of-flight (TOF) PET scanners.

  16. Development of a single multiplex amplification refractory mutation system PCR for the detection of rifampin-resistant Mycobacterium tuberculosis.

    PubMed

    Shi, Xiaodan; Zhang, Chen; Shi, Ming; Yang, Mengjie; Zhang, Yi; Wang, Ji; Shen, Hongwei; Zhao, Gang; Ma, Xuejun

    2013-11-01

    A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations.

  17. Clinical Relevance of Pathogens Detected by Multiplex PCR in Blood of Very-Low-Birth Weight Infants with Suspected Sepsis – Multicentre Study of the German Neonatal Network

    PubMed Central

    Buer, Jan; Dördelmann, Michael; Felderhoff-Müser, Ursula; Höhn, Thomas; Hepping, Nico; Hillebrand, Georg; Kribs, Angela; Marissen, Janina; Olbertz, Dirk; Rath, Peter-Michael; Schmidtke, Susanne; Siegel, Jens; Herting, Egbert; Göpel, Wolfgang

    2016-01-01

    Introduction In the German Neonatal Network (GNN) 10% of very-low-birth weight infants (VLBWI) suffer from blood-culture confirmed sepsis, while 30% of VLBWI develop clinical sepsis. Diagnosis of sepsis is a difficult task leading to potential over-treatment with antibiotics. This study aims to investigate whether the results of blood multiplex-PCR (SeptiFast®) for common sepsis pathogens are relevant for clinical decision making when sepsis is suspected in VLBWI. Methods We performed a prospective, multi-centre study within the GNN including 133 VLBWI with 214 episodes of suspected late onset sepsis (LOS). In patients with suspected sepsis a multiplex-PCR (LightCycler SeptiFast MGRADE-test®) was performed from 100 μl EDTA blood in addition to center-specific laboratory biomarkers. The attending neonatologist documented whether the PCR-result, which was available after 24 to 48 hrs, had an impact on the choice of antibiotic drugs and duration of therapy. Results PCR was positive in 110/214 episodes (51%) and blood culture (BC) was positive in 55 episodes (26%). Both methods yielded predominantly coagulase-negative staphylococci (CoNS) followed by Escherichia coli and Staphylococcus aureus. In 214 BC—PCR paired samples concordant results were documented in 126 episodes (59%; n = 32 were concordant pathogen positive results, n = 94 were negative in both methods). In 65 episodes (30%) we found positive PCR results but negative BCs, with CoNS being identified in 43 (66%) of these samples. Multiplex-PCR results influenced clinical decision making in 30% of episodes, specifically in 18% for the choice of antimicrobial therapy and in 22% for the duration of antimicrobial therapy. Conclusions Multiplex-PCR results had a moderate impact on clinical management in about one third of LOS-episodes. The main advantage of multiplex-PCR was the rapid detection of pathogens from micro-volume blood samples. In VLBWI limitations include risk of contamination, lack of resistance

  18. Multiplex PCR (polymerase chain reaction) assay for detection of E. coli O157:H7, Salmonella sp., Vibrio cholerae and Vibrio parahaemolyticus in spiked shrimps (Penaeus monodon).

    PubMed

    Fakruddin, M D; Sultana, Mahmuda; Ahmed, Monzur Morshed; Chowdhury, Abhijit; Choudhury, Naiyyum

    2013-03-15

    The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results. PMID:24498789

  19. Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens.

    PubMed

    Paradis, M-È; Haine, D; Gillespie, B; Oliver, S P; Messier, S; Comeau, J; Scholl, D T

    2012-11-01

    Bacteriological culture (BC) is the traditional method for intramammary infection diagnosis but lacks sensitivity and is time consuming. Multiplex real-time PCR (mr-PCR) enables testing the presence of several bacteria and reduces diagnosis time. Our objective was to estimate bacterial species-specific sensitivity (Se) and specificity of both BC and mr-PCR tests for detecting bacteria in milk samples from clinical mastitis cases and from apparently normal quarters, using a Bayesian latent class model. Milk samples from 1,014 clinical mastitis cases and 1,495 samples from apparently normal quarters were analyzed by BC and mr-PCR. Two positive culture definitions were used: ≥1 cfu/0.01 mL and ≥10 cfu/0.01 mL of the specified bacteria. The mr-PCR was designed to simultaneously detect Staphylococcus aureus, Streptococcus uberis, Escherichia coli, and Streptococcus agalactiae. The priors used in our Bayesian model were weakly informative, with BC priors using the best available error data. Results were compared with those obtained using uniform priors for mr-PCR to test robustness. Weak and uniform priors gave about the same posterior distributions except for Strep. uberis from normal quarters and Strep. agalactiae. Multiplex real-time PCR Se on milk from clinical mastitis were lower than mr-PCR Se on milk from normal quarters. Multiplex real-time PCR Se was higher than BC on milk from normal quarters. Multiplex real-time PCR Se was generally lower than BC on milk from clinical mastitis and it varied by clinical severity. The estimate specificities of detection for all pathogens were ≥99%, regardless of sample type. The effect of milk sample preservation before testing was evaluated and may have been a factor that affected our observed results. A significant association was observed between sample age and mr-PCR results leading to reduced detection of E. coli and Strep. agalactiae in nonclinical samples. Differences in sample age between conduct of BC and of mr-PCR

  20. Differentiation of human influenza A viruses including the pandemic subtype H1N1/2009 by conventional multiplex PCR.

    PubMed

    Furuse, Yuki; Odagiri, Takashi; Okada, Takashi; Khandaker, Irona; Shimabukuro, Kozue; Sawayama, Rumi; Suzuki, Akira; Oshitani, Hitoshi

    2010-09-01

    April 2009 witnessed the emergence of a novel H1N1 influenza A virus infecting the human population. Currently, pandemic and seasonal influenza viruses are co-circulating in human populations. Understanding the course of the emerging pandemic virus is important. It is still unknown how the novel virus co-circulates with or outcompetes seasonal viruses. Sustainable and detailed influenza surveillance is required throughout the world including developing countries. In the present study, a multiplex PCR using four primers was developed, which was designed to differentiate the pandemic H1N1 virus from the seasonal H1N1 and H3N2 viruses, to obtain amplicons of different sizes. Multiplex PCR analysis could clearly differentiate the three subtypes of human influenza A virus. This assay was performed using 206 clinical samples collected in 2009 in Japan. Between February and April, four samples were subtyped as seasonal H1N1 and four as seasonal H3N2. All samples collected after July were subtyped as pandemic H1N1. Currently, pandemic viruses seem to have replaced seasonal viruses almost completely in Japan. This is a highly sensitive method and its cost is low. Influenza surveillance using this assay would provide significant information on the epidemiology of both pandemic and seasonal influenza.

  1. Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens.

    PubMed

    Echeita, M Aurora; Herrera, Silvia; Garaizar, Javier; Usera, Miguel A

    2002-03-01

    Most Salmonella serotypes alternatively express phase 1 or phase 2 flagellar antigens encoded by fliC and fljB genes respectively. Flagellar phase reversal to identify both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fljB genes encoding H:1,w, H:e,n,x and H:e,n,z15 antigens have been sequenced and the specific sites for each antigen determined in selected Salmonella serotypes. These results, together with flagellar H1 complex variable internal sequences previously published, have been used to design a multiplex-PCR to identify H:1,2, H:1,5, H:1,6, H:1,7, H:1,w, H:e,n,x and H:e,n,z15 second-phase antigens. These antigens are part of the most common Salmonella serotypes possessing second-phase flagellar antigens. This multiplex-PCR includes 10 primers. A total of 140 Salmonella strains associated with 49 different serotypes were tested. Each strain generated one second-phase-specific antigen fragment, ranging between 50 and 400 bps. Twenty-five strains associated with 17 serotypes, with no second-phase antigen or with an antigen different from those tested in this work, did not generate any fragments. The method is quick, specific and reproducible and is independent of the phase expressed by the bacteria when tested.

  2. Investigate the correlation between clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia individually or as a “Red complex” by a multiplex PCR method

    PubMed Central

    Sanghavi, Tulsi Hasmukhrai; Shah, Nimisha; Shah, Ruchi Rani; Sanghavi, Akta

    2014-01-01

    Aim: The aim of this study was to investigate the correlation between endodontic clinical signs and symptoms and the presence of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia or their association by Multiplex polymerase chain reaction assay. Materials and Methods: Microbial samples were taken from 30 cases with necrotic pulp tissues in primary infections. DNA was extracted from the samples, which were analyzed for the presence of three endodontic pathogens by using species-specific primers. Results: P. gingivalis, T. denticola, T. forsythia, and Red Complex were present in 11, 17, 4, and 2 canals, respectively. Clinical and statistically significant relationships were found between T. forsythia and mobility and between T. denticola and swelling. (P < 0.05). Presence of other Red complex bacteria shows clinical association with presence of signs and symptoms but no statistically significant relationship. Conclusion: The high prevalence of P. gingivalis, T. denticola, and T. forsythia in the examined samples suggests that these bacteria are related to the etiology of symptomatic periradicular diseases. PMID:25506144

  3. The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus.

    PubMed

    Dung, Tran Thi Ngoc; Phat, Voong Vinh; Nga, Tran Vu Thieu; My, Phan Vu Tra; Duy, Pham Thanh; Campbell, James I; Thuy, Cao Thu; Hoang, Nguyen Van Minh; Van Minh, Pham; Le Phuc, Hoang; Tuyet, Pham Thi Ngoc; Vinh, Ha; Kien, Duong Thi Hue; Huy, Huynh Le Anh; Vinh, Nguyen Thanh; Nga, Tran Thi Thu; Hau, Nguyen Thi Thu; Chinh, Nguyen Tran; Thuong, Tang Chi; Tuan, Ha Manh; Simmons, Cameron; Farrar, Jeremy J; Baker, Stephen

    2013-01-01

    Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. PMID:23046990

  4. Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

    PubMed Central

    Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J.; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O’Brien, Mary; Turner, Nicholas C.; Popat, Sanjay; Downward, Julian

    2015-01-01

    Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

  5. Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

    PubMed

    Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O'Brien, Mary; Turner, Nicholas C; Popat, Sanjay; Downward, Julian

    2015-01-01

    Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

  6. Autonomous Detection of Aerosolized Biological Agents by Multiplexed Immunoassay with PCR Confirmation

    SciTech Connect

    Hindson, B J; McBride, M T; Makarewicz, A J; Henderer, B D; Setlur, U S; Smith, S M; Gutierrez, D M; Metz, T R; Nasarabadi, S L; Venkateswaran, K S; Farrow, S W; Colston, Jr., B W; Dzenitis, J M

    2004-05-27

    The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii and botulinum toxoid. By coupling highly selective antibody and DNA based assays, the probability of an APDS reporting a false positive is extremely low.

  7. Single-channel multiplexing without melting curve analysis in real-time PCR.

    PubMed

    Lee, Young-Jo; Kim, Daeyoung; Lee, Kihoon; Chun, Jong-Yoon

    2014-01-01

    Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics. PMID:25501038

  8. Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9.

    PubMed

    Zhang, Yanjun; Mao, Haiyan; Yan, Juying; Wang, Xinying; Zhang, Lei; Guus, Koch; Li, Hui; Li, Zhen; Chen, Yin; Gong, Liming; Chen, Zhiping; Xia, Shichang

    2014-07-01

    Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10(-1) TCID50/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.

  9. A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.).

    PubMed

    Biswas, C; Dey, P; Satpathy, S

    2013-05-01

    A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C.

  10. A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.).

    PubMed

    Biswas, C; Dey, P; Satpathy, S

    2013-05-01

    A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C. PMID:23413927

  11. Rapid and Sensitive PCR-Dipstick DNA Chromatography for Multiplex Analysis of the Oral Microbiota

    PubMed Central

    Niwa, Kousuke; Kawase, Mitsuo; Tanner, Anne C. R.; Takahashi, Nobuhiro

    2014-01-01

    A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases. PMID:25485279

  12. Detection and Identification of Actinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

    PubMed Central

    Lo, Terry M.; Ward, Christine K.; Inzana, Thomas J.

    1998-01-01

    Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 102 CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay. PMID:9620404

  13. Simultaneous detection and differentiation of four closely related sweet potato potyviruses by a multiplex one-step RT-PCR.

    PubMed

    Li, Fan; Zuo, Ruijuan; Abad, Jorge; Xu, Donglin; Bao, Gaili; Li, Ruhui

    2012-12-01

    Four closely related potyviruses, Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG) and/or Sweet potato virus 2 (SPV2), are involved in sweet potato virus disease complexes worldwide. Identification and detection of these viruses are complicated by high similarity among their genomic sequences, frequent occurrence as mixed infections and low titer in many sweet potato cultivars. A one-tube multiplex reverse transcription-PCR (mRT-PCR) assay was developed for simultaneous detection and differentiation of SPFMV, SPVC, SPVG and SPV2. Four specific forward primers unique to each virus and one reverse primer based on the region conserved in all four viruses were selected and used in the assay. The mRT-PCR assay was optimized for primer concentration and cycling conditions. It was tested using sweet potato plants infected naturally with one to four target viruses and then evaluated using field samples collected from southwestern China. The mRT-PCR assay is reliable and sensitive as a simple, rapid and cost-effective method to detect these pathogens in sweet potato. This assay will be useful to quarantine and certification programs and virus surveys when large numbers of samples are tested.

  14. A Low-Cost Efficient Multiplex PCR for Prenatal Sex Determination in Bovine Fetus Using Free Fetal DNA in Maternal Plasma

    PubMed Central

    Davoudi, Arash; Seighalani, Ramin; Aleyasin, Seyed Ahmad; Tarang, Alireza; Salehi, Abdolreza Salehi; Tahmoressi, Farideh

    2012-01-01

    Background In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction (PCR) analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. Materials and Methods In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 µl maternal plasma. Two primer pairs for bovine amelogenin gene (bAML) and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. Results The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. Conclusion The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses. PMID:25505511

  15. Multiplex real-time PCR monitoring of intestinal helminths in humans reveals widespread polyparasitism in Northern Samar, the Philippines.

    PubMed

    Gordon, Catherine A; McManus, Donald P; Acosta, Luz P; Olveda, Remigio M; Williams, Gail M; Ross, Allen G; Gray, Darren J; Gobert, Geoffrey N

    2015-06-01

    The global socioeconomic importance of helminth parasitic disease is underpinned by the considerable clinical impact on millions of people. While helminth polyparasitism is considered common in the Philippines, little has been done to survey its extent in endemic communities. High morphological similarity of eggs between related species complicates conventional microscopic diagnostic methods which are known to lack sensitivity, particularly in low intensity infections. Multiplex quantitative PCR diagnostic methods can provide rapid, simultaneous identification of multiple helminth species from a single stool sample. We describe a multiplex assay for the differentiation of Ascaris lumbricoides, Necator americanus, Ancylostoma, Taenia saginata and Taenia solium, building on our previously published findings for Schistosoma japonicum. Of 545 human faecal samples examined, 46.6% were positive for at least three different parasite species. High prevalences of S. japonicum (90.64%), A. lumbricoides (58.17%), T. saginata (42.57%) and A. duodenale (48.07%) were recorded. Neither T. solium nor N. americanus were found to be present. The utility of molecular diagnostic methods for monitoring helminth parasite prevalence provides new information on the extent of polyparasitism in the Philippines municipality of Palapag. These methods and findings have potential global implications for the monitoring of neglected tropical diseases and control measures.

  16. Multiplex real-time PCR monitoring of intestinal helminths in humans reveals widespread polyparasitism in Northern Samar, the Philippines.

    PubMed

    Gordon, Catherine A; McManus, Donald P; Acosta, Luz P; Olveda, Remigio M; Williams, Gail M; Ross, Allen G; Gray, Darren J; Gobert, Geoffrey N

    2015-06-01

    The global socioeconomic importance of helminth parasitic disease is underpinned by the considerable clinical impact on millions of people. While helminth polyparasitism is considered common in the Philippines, little has been done to survey its extent in endemic communities. High morphological similarity of eggs between related species complicates conventional microscopic diagnostic methods which are known to lack sensitivity, particularly in low intensity infections. Multiplex quantitative PCR diagnostic methods can provide rapid, simultaneous identification of multiple helminth species from a single stool sample. We describe a multiplex assay for the differentiation of Ascaris lumbricoides, Necator americanus, Ancylostoma, Taenia saginata and Taenia solium, building on our previously published findings for Schistosoma japonicum. Of 545 human faecal samples examined, 46.6% were positive for at least three different parasite species. High prevalences of S. japonicum (90.64%), A. lumbricoides (58.17%), T. saginata (42.57%) and A. duodenale (48.07%) were recorded. Neither T. solium nor N. americanus were found to be present. The utility of molecular diagnostic methods for monitoring helminth parasite prevalence provides new information on the extent of polyparasitism in the Philippines municipality of Palapag. These methods and findings have potential global implications for the monitoring of neglected tropical diseases and control measures. PMID:25858090

  17. Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

    PubMed

    Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; P<0.001), 100 % (95 % CI, 0.983-1.000; P<0.001), 100 % and 99 %, respectively. However, positivity of the Real-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

  18. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

    PubMed Central

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-01-01

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets. PMID:27739510

  19. Virulotyping of Salmonella enterica subsp. enterica isolated from indigenous vegetables and poultry meat in Malaysia using multiplex-PCR.

    PubMed

    Khoo, Chai-Hoon; Cheah, Yoke-Kqueen; Lee, Learn-Han; Sim, Jiun-Horng; Salleh, Noorzaleha Awang; Sidik, Shiran Mohd; Radu, Son; Sukardi, Sabrina

    2009-11-01

    The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70% of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of DNA 0.8 pg microl(-1). This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies.

  20. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

    NASA Astrophysics Data System (ADS)

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-10-01

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

  1. [Development, optimization and application of the expression analysis platform based on multiplex quantitative RT-PCR using fluorescent universal primers].

    PubMed

    Wang, Qin-Xi; Li, Kai; Zhou, Yu-Xun; Xiao, Jun-Hua

    2009-05-01

    A multiplex quantitative RT-PCR technology with a universal fluorescent primer was established. This technology employs a chimeric-primer-induced-universal-primer amplification method that ensures target genes amplified in a constant ratio. This technique was cost-effective, moderate-throughput, and reliable in quantification of gene expression. It is complementary to cDNA chip, which has low quantitative accuracy , and Real-time quantitative PCR with low throughput, through improving the entire process of expression profiling analysis. Eleven genes within a QTL segment regulating mouse puberty onset on chromosome X were investigated to construct and optimize the method. The sensitivity of detection (102 copies) was determined, the concentration ratio of universal primer and chimeric forward primers (1:1) was optimized, and the accuracy and repeatability were validated. The method of Touchdown PCR with addition of universal primers significantly improved amplification of genes expressed in low abundance. After testing the expression profile of 11 genes in hypothalamus and testis in two mouse strains C3H/HeJ and C57BL/6J at the age of 15 d, one gene named PHF6 was found differentially expressed for further function analysis.

  2. Computing TaqMan probes for multiplex PCR detection of E. coli O157 serotypes in water.

    PubMed

    Ram, Siya; Shanker, Rishi

    2005-01-01

    Diarrheagenic E. coli strains contribute to water related diseases in urban and rural environment in developing and developed world. E. coli pathotype and pathogenicity varies due to complex multifactorial mechanism involving a large number of virulence factors. Rapid assessment of the virulence pattern of E. coli isolates is possible by Real-Time PCR probes like TaqMan. For designing TaqMan probes and primers for multiplex PCR selected E. coli gene sequences: stx1, stx2, hlyA, chuA, eae, lacZ, lamB and fimA were retrieved from NCBI's GenBank database. The alignment of the multiple sequences and analysis of conserved sequences was carried out using ClustalW and BLAST programs. The primers and Taqmen probes were designed using Beacon Designer software version 2.1 for two multiplexed PCR assays. In silico PCR simulation of these assays showed PCR products for stx2 (248bp) stx1 (102 bp), lacZ (228bp) and lamB (86 bp) in multiplex #1 and eae (200bp), chuA (147 bp), hlyA (141bp) and fimA (79 bp) in multiplex #2, respectively. These multiplexed PCR amplification products and probes can be used to identify and confirm presence of O157:H7/ H7-, O157:H43/45 and O26:H-/H11 serotypes. In conclusion, multiplex Real-Time Polymerase Chain Reaction oligomers and TaqMan probes designed and validated in silico will be helpful in management of water quality and outbreaks, by improving specificity and minimizing time needed for in vitro verification work.

  3. Group-Specific Multiplex PCR Detection Systems for the Identification of Flying Insect Prey

    PubMed Central

    Sint, Daniela; Niederklapfer, Bettina; Kaufmann, Ruediger; Traugott, Michael

    2014-01-01

    The applicability of species-specific primers to study feeding interactions is restricted to those ecosystems where the targeted prey species occur. Therefore, group-specific primer pairs, targeting higher taxonomic levels, are often desired to investigate interactions in a range of habitats that do not share the same species but the same groups of prey. Such primers are also valuable to study the diet of generalist predators when next generation sequencing approaches cannot be applied beneficially. Moreover, due to the large range of prey consumed by generalists, it is impossible to investigate the breadth of their diet with species-specific primers, even if multiplexing them. However, only few group-specific primers are available to date and important groups of prey such as flying insects have rarely been targeted. Our aim was to fill this gap and develop group-specific primers suitable to detect and identify the DNA of common taxa of flying insects. The primers were combined in two multiplex PCR systems, which allow a time- and cost-effective screening of samples for DNA of the dipteran subsection Calyptratae (including Anthomyiidae, Calliphoridae, Muscidae), other common dipteran families (Phoridae, Syrphidae, Bibionidae, Chironomidae, Sciaridae, Tipulidae), three orders of flying insects (Hymenoptera, Lepidoptera, Plecoptera) and coniferous aphids within the genus Cinara. The two PCR assays were highly specific and sensitive and their suitability to detect prey was confirmed by testing field-collected dietary samples from arthropods and vertebrates. The PCR assays presented here allow targeting prey at higher taxonomic levels such as family or order and therefore improve our ability to assess (trophic) interactions with flying insects in terrestrial and aquatic habitats. PMID:25525799

  4. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.

  5. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. PMID:27506582

  6. Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay.

    PubMed

    Maksyutov, Rinat A; Gavrilova, Elena V; Shchelkunov, Sergei N

    2016-10-01

    A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species.

  7. Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay.

    PubMed

    Maksyutov, Rinat A; Gavrilova, Elena V; Shchelkunov, Sergei N

    2016-10-01

    A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species. PMID:27477914

  8. A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

    PubMed

    Boubaker, Ghalia; Macchiaroli, Natalia; Prada, Laura; Cucher, Marcela A; Rosenzvit, Mara C; Ziadinov, Iskender; Deplazes, Peter; Saarma, Urmas; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2013-01-01

    Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

  9. Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

    PubMed Central

    Perera, Piyumali K.; Gasser, Robin B.; Firestone, Simon M.; Smith, Lee; Roeber, Florian

    2014-01-01

    Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. PMID:25339402

  10. Rapid Detection of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in Fresh Chicken Meat and By-Products in Bangkok, Thailand, Using Modified Multiplex PCR.

    PubMed

    Saiyudthong, S; Phusri, K; Buates, S

    2015-07-01

    A multiplex PCR assay for simultaneous detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari was developed and validated to assess the occurrence of these bacteria in fresh chicken meat and by-products in Bangkok, Thailand, by using a new combination of four previously published PCR primers for C. jejuni, C. coli, C. lari, and a universal 16S rDNA gene as an internal control. The specificity was determined by using 13 strains of other bacteria. With pure culture DNA, the detection limit was 0.017 ng/PCR for C. jejuni and C. coli and was 0.016 ng/PCR for C. lari. It can detect 10 CFU of C. jejuni, C. coli, and C. lari in 2 g of chicken meat within a 16-h enrichment time. Our multiplex PCR assay was applied for identification of Campylobacter spp. in 122 supermarket samples and 108 fresh market samples. Of the 230 samples evaluated by multiplex PCR, 54.0, 3.3, and 10.7% of supermarket samples were positive for C. jejuni, C. coli, and mixed C. jejuni and C. coli, respectively, and 56.5 and 33.3% of fresh market samples were positive for C. jejuni and mixed C. jejuni and C. coli, respectively. No sample was positive for C. lari. Fresh market samples had significantly higher C. jejuni and C. coli contamination than those from supermarkets (relative risk: 1.3; P = 0.0001). Compared with the culture method (a gold standard), the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of multiplex PCR were 97.7, 86.8, 96.1, 92.0, and 95.2%, respectively. No significant difference was observed between results from two methods (P = 0.55). Therefore, the established multiplex PCR was not only rapid and easy to perform but had a high sensitivity and specificity to distinguish between C. jejuni, C. coli, and C. lari, even in samples containing mixed contamination. Our study indicated that fresh chicken meat and by-products from fresh markets were significantly less hygienic than those

  11. A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens.

    PubMed

    Hughesman, Curtis B; Lu, X J David; Liu, Kelly Y P; Zhu, Yuqi; Poh, Catherine F; Haynes, Charles

    2016-01-01

    The ability of droplet digital PCR (ddPCR) to accurately determine the concentrations of amplifiable targets makes it a promising platform for measuring copy number alterations (CNAs) in genomic biomarkers. However, its application to clinical samples, particularly formalin-fixed paraffin-embedded specimens, will require strategies to reliably determine CNAs in DNA of limited quantity and quality. When applied to cancerous tissue, those methods must also account for global genetic instability and the associated probability that the abundance(s) of one or more chosen reference loci do not represent the average ploidy of cells comprising the specimen. Here we present an experimental design strategy and associated data analysis tool that enables accurate determination of CNAs in a panel of biomarkers using multiplexed ddPCR. The method includes strategies to optimize primer and probes design to cleanly segregate droplets in the data output from reaction wells amplifying multiple independent templates, and to correct for bias from artifacts such as DNA fragmentation. We demonstrate how a panel of reference loci can be used to determine a stable CNA-neutral benchmark. These innovations, when taken together, provide a comprehensive strategy that can be used to reliably detect biomarker CNAs in DNA extracted from either frozen or FFPE tissue biopsies.

  12. A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens

    PubMed Central

    Hughesman, Curtis B.; Lu, X. J. David; Liu, Kelly Y. P.; Zhu, Yuqi; Poh, Catherine F.; Haynes, Charles

    2016-01-01

    The ability of droplet digital PCR (ddPCR) to accurately determine the concentrations of amplifiable targets makes it a promising platform for measuring copy number alterations (CNAs) in genomic biomarkers. However, its application to clinical samples, particularly formalin-fixed paraffin-embedded specimens, will require strategies to reliably determine CNAs in DNA of limited quantity and quality. When applied to cancerous tissue, those methods must also account for global genetic instability and the associated probability that the abundance(s) of one or more chosen reference loci do not represent the average ploidy of cells comprising the specimen. Here we present an experimental design strategy and associated data analysis tool that enables accurate determination of CNAs in a panel of biomarkers using multiplexed ddPCR. The method includes strategies to optimize primer and probes design to cleanly segregate droplets in the data output from reaction wells amplifying multiple independent templates, and to correct for bias from artifacts such as DNA fragmentation. We demonstrate how a panel of reference loci can be used to determine a stable CNA-neutral benchmark. These innovations, when taken together, provide a comprehensive strategy that can be used to reliably detect biomarker CNAs in DNA extracted from either frozen or FFPE tissue biopsies. PMID:27537682

  13. A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens.

    PubMed

    Hughesman, Curtis B; Lu, X J David; Liu, Kelly Y P; Zhu, Yuqi; Poh, Catherine F; Haynes, Charles

    2016-01-01

    The ability of droplet digital PCR (ddPCR) to accurately determine the concentrations of amplifiable targets makes it a promising platform for measuring copy number alterations (CNAs) in genomic biomarkers. However, its application to clinical samples, particularly formalin-fixed paraffin-embedded specimens, will require strategies to reliably determine CNAs in DNA of limited quantity and quality. When applied to cancerous tissue, those methods must also account for global genetic instability and the associated probability that the abundance(s) of one or more chosen reference loci do not represent the average ploidy of cells comprising the specimen. Here we present an experimental design strategy and associated data analysis tool that enables accurate determination of CNAs in a panel of biomarkers using multiplexed ddPCR. The method includes strategies to optimize primer and probes design to cleanly segregate droplets in the data output from reaction wells amplifying multiple independent templates, and to correct for bias from artifacts such as DNA fragmentation. We demonstrate how a panel of reference loci can be used to determine a stable CNA-neutral benchmark. These innovations, when taken together, provide a comprehensive strategy that can be used to reliably detect biomarker CNAs in DNA extracted from either frozen or FFPE tissue biopsies. PMID:27537682

  14. A novel multiplex-PCR for the rapid identification of Mycobacterium bovis in clinical isolates of both veterinary and human origin.

    PubMed Central

    Cobos-Marín, L.; Montes-Vargas, J.; Rivera-Gutierrez, S.; Licea-Navarro, A.; González-y-Merchand, J. A.; Estrada-García, I.

    2003-01-01

    Bovine tuberculosis is a zoonotic disease that not only causes huge economic losses but also poses an important risk for human infection. The definitive identification of a clinical isolate relies on time-consuming, highly specialized and laborious biochemical tests. We have developed a method for the rapid and reliable identification of Mycobacterium bovis and for its simultaneous differentiation from other members of the M. tuberculosis complex. Furthermore, the technique also allowed us to distinguish M. tuberculosis complex members from other Mycobacterial species. The method comprises both a single PCR and a multiplex-PCR and can be confidently applied to samples of both veterinary and human origin. PMID:12825733

  15. Development of Multiplex RT-PCR for Simultaneous Detection of Garlic Viruses and the Incidence of Garlic Viral Disease in Garlic Genetic Resources

    PubMed Central

    Nam, Moon; Lee, Yeong-Hoon; Park, Chung Youl; Lee, Min-A; Bae, Yang-Soo; Lim, Seungmo; Lee, Joong Hwan; Moon, Jae Sun; Lee, Su-Heon

    2015-01-01

    Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic. PMID:25774116

  16. Development of a multiplex real time PCR to detect thermophilic lactic acid bacteria in natural whey starters.

    PubMed

    Bottari, Benedetta; Agrimonti, Caterina; Gatti, Monica; Neviani, Erasmo; Marmiroli, Nelson

    2013-01-01

    A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of

  17. Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs).

    PubMed

    Dörries, Hans-Henno; Remus, Ivonne; Grönewald, Astrid; Grönewald, Cordt; Berghof-Jäger, Kornelia

    2010-03-01

    The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.

  18. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics. PMID:25746154

  19. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics.

  20. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays.

    PubMed

    Inderbitzin, Patrik; Davis, R Michael; Bostock, Richard M; Subbarao, Krishna V

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.

  1. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

    PubMed Central

    Inderbitzin, Patrik; Davis, R. Michael; Bostock, Richard M.; Subbarao, Krishna V.

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers. PMID:23823707

  2. A duplicated PLP gene causing Pelizaeus-Merzbacher disease detected by comparative multiplex PCR.

    PubMed Central

    Inoue, K.; Osaka, H.; Sugiyama, N.; Kawanishi, C.; Onishi, H.; Nezu, A.; Kimura, K.; Yamada, Y.; Kosaka, K.

    1996-01-01

    Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused by abnormalities in the proteolipid protein (PLP) gene, which is essential for oligodendrocyte differentiation and CNS myelin formation. Although linkage analysis has shown the homogeneity at the PLP locus in patients with PMD, exonic mutations in the PLP gene have been identified in only 10%-25% of all cases, which suggests the presence of other genetic aberrations, including gene duplication. In this study, we examined five families with PMD not carrying exonic mutations in PLP gene, using comparative multiplex PCR (CM-PCR) as a semiquantitative assay of gene dosage. PLP gene duplications were identified in four families by CM-PCR and confirmed in three families by densitometric RFLP analysis. Because a homologous myelin protein gene, PMP22, is duplicated in the majority of patients with Charcot-Marie-Tooth 1A, PLP gene overdosage may be a important genetic abnormality in PMD and affect myelin formation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8659540

  3. Comparison of staining techniques and multiplex nested PCR for diagnosis of intestinal microsporidiosis.

    PubMed

    Saigal, Karnika; Khurana, Sumeeta; Sharma, Aman; Sehgal, Rakesh; Malla, Nancy

    2013-11-01

    Microsporidiosis is increasingly being recognized as the cause for diarrhea in immunocompromised patients. The 2 most common microsporidia causing gastrointestinal infection worldwide are Enterocytozoon bieneusi and Encephalitozoon intestinalis. The aim of present study was to evaluate different techniques for detection of intestinal microsporidia in human stool samples. The fecal samples of 395 individuals including 125 HIV-seropositive patients with diarrhoea, 158 HIV-seropositive patients without diarrhoea, 55 HIV-seronegative patients with diarrhoea, and 57 healthy controls were used for detection of microsporidia by modified trichrome staining, calcofluor staining, and multiplex polymerase chain reaction (PCR). PCR had the highest sensitivity of 100%, while its specificity was 97.9%. Trichrome staining had highest specificity of 100% but a sensitivity of 63.8% only, and calcofluor white had a sensitivity and specificity of 79.7% and 82.2%, respectively. Thus, for diagnosis of intestinal microsporidiosis, it is important to perform PCR as staining techniques are not good enough to detect microsporidia in stool samples and for their species identification.

  4. A duplicated PLP gene causing Pelizaeus-Merzbacher disease detected by comparative multiplex PCR

    SciTech Connect

    Inoue, K.; Sugiyama, N.; Kawanishi, C.

    1996-07-01

    Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused by abnormalities in the proteolipid protein (PLP) gene, which is essential for oligodendrocyte differentiation and CNS myelin formation. Although linkage analysis has shown the homogeneity at the PLP locus in patients with PMD, exonic mutations in the PLP gene have been identified in only 10% - 25% of all cases, which suggests the presence of other genetic aberrations, including gene duplication. In this study, we examined five families with PMD not carrying exonic mutations in PLP gene, using comparative multiplex PCR (CM-PCR) as a semiquantitative assay of gene dosage. PLP gene duplications were identified in four families by CM-PCR and confirmed in three families by densitometric RFLP analysis. Because a homologous myelin protein gene, PMP22, is duplicated in the majority of patients with Charcot-Marie-Tooth 1A, PLP gene overdosage may be an important genetic abnormality in PMD and affect myelin formation. 38 ref., 5 figs., 2 tabs.

  5. DOUBLE-STEP MULTIPLEX REAL TIME PCR WITH MELTING CURVE ANALYSIS FOR DETECTION AND DIFFERENTIATION OF MYCOBACTERIA IN SPUTUM.

    PubMed

    Kasa, Sawinee; Faksri, Kiatichai; Kaewkes, Wanlop; Lulitanond, Viraphong; Namwat, Wises

    2015-01-01

    Mycobacterium tuberculosis (M. tb) is a causative agent of tuberculosis, a worldwide public health problem. In recent years, the incidence of human mycobacterial infection due to species other than M. tb has increased. However, the lack of specific, rapid, and inexpensive methods for identification of mycobacterial species remains a pressing problem. A diagnostic test was developed for mycobacterial strain differentiation utilizing a double-step multiplex real time PCR together with melting curve analysis for identifying and distinguishing among M. tb, M. bovis BCG, other members of M. tb. complex, M. avium, and non-tuberculosis mycobacteria. The assay was tested using 167 clinical sputum samples in comparison with acid-fast staining and culturing. Using only the first step (step A) the assay achieved sensitivity and specificity of 81% and 95%, respectively. The detection limit was equivalent to 50 genome copies.

  6. DOUBLE-STEP MULTIPLEX REAL TIME PCR WITH MELTING CURVE ANALYSIS FOR DETECTION AND DIFFERENTIATION OF MYCOBACTERIA IN SPUTUM.

    PubMed

    Kasa, Sawinee; Faksri, Kiatichai; Kaewkes, Wanlop; Lulitanond, Viraphong; Namwat, Wises

    2015-01-01

    Mycobacterium tuberculosis (M. tb) is a causative agent of tuberculosis, a worldwide public health problem. In recent years, the incidence of human mycobacterial infection due to species other than M. tb has increased. However, the lack of specific, rapid, and inexpensive methods for identification of mycobacterial species remains a pressing problem. A diagnostic test was developed for mycobacterial strain differentiation utilizing a double-step multiplex real time PCR together with melting curve analysis for identifying and distinguishing among M. tb, M. bovis BCG, other members of M. tb. complex, M. avium, and non-tuberculosis mycobacteria. The assay was tested using 167 clinical sputum samples in comparison with acid-fast staining and culturing. Using only the first step (step A) the assay achieved sensitivity and specificity of 81% and 95%, respectively. The detection limit was equivalent to 50 genome copies. PMID:26513906

  7. First Evaluation of an Outbreak of Bovine Babesiosis and Anaplasmosis in Southern Brazil Using Multiplex PCR

    PubMed Central

    Canever, Mariana Feltrin; Vieira, Luisa Lemos; Reck, Carolina; Richter, Luisa

    2014-01-01

    Outbreaks of tick-borne disease cases in Santa Catarina, Brazil are known, but the presence of the pathogen DNA has never been determined. In this study, the first survey of Anaplasma marginale, Babesia bigemina, and Babesia bovis DNA on blood samples of 33 cattle from an outbreak in Ponte Alta Municipality, Santa Catarina, Brazil, has been carried out. A multiplex PCR detected 54.5% of animals were co-infected with 2 or 3 parasites, while 24.2% were infected with only 1 species. The most prevalent agent was B. bigemina (63.6%) followed by A. marginale (60.6%). This is the first report of tick-borne disease pathogens obtained by DNA analysis in Southern Brazil. PMID:25352699

  8. Evaluation of degradation in DNA from males with a quantitative gender typing, endpoint PCR multiplex.

    PubMed

    Smith, Byron C; Vandegrift, Emily; Fuller, Valerie Mattimore; Allen, Robert W

    2015-03-01

    Evidentiary samples submitted to a forensic DNA laboratory occasionally yield DNA that is degraded. Samples of intact chromosomal DNA (both nuclear and mitochondrial) were subjected to a heating protocol to induce DNA degradation. The DNAs were then analyzed using a multiplex PCR assay that amplifies targets of low and high molecular weight on the X/Y and mitochondrial chromosomes. If degradation is random, the amplification of larger DNA targets should be more adversely affected by degradation than smaller targets. In nuclear and mitochondrial DNA from a male donor, exhibiting degradation, DNA quantity estimates based upon higher molecular weight amplicons (HMW) are significantly lower than estimates made using low molecular weight (LMW) Q-TAT amplicons. DNA degradation estimated using this approach correlated well with actual fluorescence associated with HMW and LMW STR alleles amplified from the same genomic DNA templates. Q-TAT is thus useful not only as a quantitation tool, but also as an indicator of template degradation.

  9. mRNA profiling for body fluid identification by multiplex quantitative RT-PCR.

    PubMed

    Juusola, Jane; Ballantyne, Jack

    2007-11-01

    An alternative approach to conventional protein-based body fluid identification is gene expression profiling analysis. In the present work, we report the development of sensitive and robust multiplex quantitative reverse transcriptase-PCR assays for the identification of blood, saliva, semen, and menstrual blood. Each body fluid assay comprises a triplex system that detects transcripts from two body fluid-specific genes and a housekeeping gene GAPDH. The body fluid-specific genes include erythroid delta-aminolevulinate synthase (ALAS2) and beta-spectrin (SPTB) for blood, statherin (STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1) and protamine 2 (PRM2) for semen, and matrix metalloproteinase 7 (MMP7) and matrix metalloproteinase 10 (MMP10) for menstrual blood. Normalization of both body fluid-specific genes to the housekeeping gene by means of appropriate cycle threshold metrics ensures the high specificity of each assay for its cognate body fluid.

  10. Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods.

    PubMed

    Ali, Md Eaqub; Razzak, Md Abdur; Hamid, Sharifah Bee Abd; Rahman, Md Mahfujur; Amin, Md Al; Rashid, Nur Raifana Abd; Asing

    2015-06-15

    Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation.

  11. Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification.

    PubMed

    Park, Seong-Min; Park, Seong-Yeon; Kim, Jeong-Hwan; Kang, Tae-Wook; Park, Jong-Lyul; Woo, Kwang-Man; Kim, Jong-Sik; Lee, Han-Chul; Kim, Seon-Young; Lee, Seung-Hwan

    2013-01-01

    In forensic science, identifying a tissue where a forensic specimen was originated is one of the principal challenges. Messenger RNA (mRNA) profile clearly reveals tissue-specific gene expression patterns that many attempts have been made to use RNA for forensic tissue identification. To systematically investigate the body-fluid-specific expression of mRNAs and find novel mRNA markers for forensic body fluid identification, we performed DNA microarray experiment with 24 Korean body fluid samples. Shannon entropy and Q-values were calculated for each gene, and 137 body-fluid-specific candidate genes were selected. By applying more stringent criteria, we further selected 28 candidate genes and validated them by RT-PCR and qRT-PCR. As a result, we suggest a novel combination of four body-fluid-specific mRNA makers: PPBP for blood, FDCSP for saliva, MSMB for semen and MSLN for vaginal secretion. Multiplex qRT-PCR assay was designed using the four mRNA markers and DNA/RNA co-extraction method was tested for forensic use. This study will provide a thorough examination of body-fluid-specifically expressed mRNAs, which will enlarge the possibility of practical use of RNA for forensic purpose.

  12. A spot multiplex nested RT-PCR for the simultaneous and generic detection of viruses involved in the aetiology of grapevine leafroll and rugose wood of grapevine.

    PubMed

    Dovas, C I; Katis, N I

    2003-05-01

    A spot nested RT-PCR assay using degenerate deoxyinosine-containing primers was developed, allowing rapid and simultaneous detection of Closterovirus sequences. Nested PCR amplification increased the specificity and sensitivity of detection. The sensitivity was also increased by a factor of 10 by using in addition to the deoxyinosine (dI)-containing primers, respective homologous primers in which dI was substituted by dG in the region of sequence homology. These homologous primers are shorter, having lower degeneracy and higher amplification efficiency than the dI-containing primers. This method was coupled to a similar nested RT-PCR detection method for Vitivirus and Foveavirus sequences. This permitted multiplex RT-PCR amplification of sequences belonging to the three genera in the same reaction tube and the two subsequent nested PCR amplifications (one for closteroviruses and one for viti- and foveaviruses) to run in parallel. Different primers and amplification parameters (additives and thermocycling conditions) were evaluated and optimised, respectively, in order to amplify efficiently all different templates. These improvements permitted the multiplex detection of fovea- and closteroviruses in petiole and cortical scraping preparations from 23 infected field-grown grapevines throughout the year, with the exception of GLRaV-1 in petioles that was only possible from June onwards. Preliminary results show that this method can detect reliably virus species from three genera in grapevine allowing simple, fast and cost-effective testing of a large number of samples in certification schemes.

  13. Capsular typing of Streptococcus pneumoniae isolated in an Algerian hospital using a new multiplex PCR-based scheme.

    PubMed

    Ziane, Hanifa; Manageiro, Vera; Ferreira, Eugénia; Bektache, Soumia; Tazir, Mohamed; Caniça, Manuela

    2015-12-01

    We developed a new sequential multiplex-PCR-based typing scheme (MPBTS) for pneumococcal capsular classification. The serogroup/type of 37 control isolates obtained by the Quellung reaction, MPBTS, and nucleotide sequencing, were fully concordant. The serogroups/types of 75 invasive isolates determined by MPBTS, presented 100% specificity and 96% sensitivity, when compared with the Quellung reaction. PMID:26546733

  14. A multiplex PCR assay for determination of mating type in isolates of the honey bee fungal pathogen, Ascosphaera apis.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we developed a multiplex PCR for identification of mating type idiomorphs in the filamentous fungus, Ascosphaera apis, the causative agent of chalkbrood disease in the honey bee (Apis melliffera). A combination of gene-specific primers was designed to amplify Mat1-1 and Mat1-2 gene fra...

  15. Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to determine the genetic basis for loss of fumonisin B¬2 (FB2) biosynthesis in FB2 non-producing A. niger strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified in FB2-produci...

  16. Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products

    PubMed Central

    Pelisser, Marcia Regina; Klein, Cátia Silene; Ascoli, Kelen Regina; Zotti, Thaís Regina; Arisi, Ana Carolina Maisonnave

    2009-01-01

    Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see. PMID:24031334

  17. One-Step Multiplex PCR Assay for Differentiating Proposed New Species “Clostridium neonatale” from Closely Related Species

    PubMed Central

    Ferraris, Laurent; Schönherr, Sophia; Bouvet, Philippe; Dauphin, Brunhilde; Popoff, Michel; Butel, Marie Jose

    2015-01-01

    “Clostridium neonatale” sp. nov., previously involved in an outbreak of neonatal necrotizing enterocolitis, was recently proposed as a new species of the Clostridium genus sensu stricto. We developed a one-step multiplex colony PCR for C. neonatale identification and investigated C. neonatale intestinal colonization frequency in healthy preterm neonates. PMID:26292306

  18. Capsular typing of Streptococcus pneumoniae isolated in an Algerian hospital using a new multiplex PCR-based scheme.

    PubMed

    Ziane, Hanifa; Manageiro, Vera; Ferreira, Eugénia; Bektache, Soumia; Tazir, Mohamed; Caniça, Manuela

    2015-12-01

    We developed a new sequential multiplex-PCR-based typing scheme (MPBTS) for pneumococcal capsular classification. The serogroup/type of 37 control isolates obtained by the Quellung reaction, MPBTS, and nucleotide sequencing, were fully concordant. The serogroups/types of 75 invasive isolates determined by MPBTS, presented 100% specificity and 96% sensitivity, when compared with the Quellung reaction.

  19. Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR.

    PubMed

    Basuni, M; Mohamed, Z; Ahmad, M; Zakaria, N Z; Noordin, R

    2012-09-01

    Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.

  20. Multiplex RT-PCR assay for the differential diagnosis of alveolar rhabdomyosarcoma and Ewing's sarcoma.

    PubMed Central

    Downing, J. R.; Khandekar, A.; Shurtleff, S. A.; Head, D. R.; Parham, D. M.; Webber, B. L.; Pappo, A. S.; Hulshof, M. G.; Conn, W. P.; Shapiro, D. N.

    1995-01-01

    Cytogenetic analysis has defined specific translocations associated with two of the most common small round cell tumors of childhood, t(11;22) in Ewing's sarcoma and t(2;13) in alveolar rhabdomyosarcoma. We and others have previously demonstrated the diagnostic utility of a reverse transcriptase polymerase chain reaction (RT-PCR) assay for the detection of the t(11;22) encoded EWS/FLI-1 chimeric message in Ewing's sarcoma. More recently, we have cloned the t(2;13)(q35;q14) translocation and have shown that it results in the fusion of the PAX3 gene on chromosome 2 to FKHR, a novel member of the fork-head family of transcription factors on chromosome 13. To define the morphological spectrum of childhood sarcomas that express the t(2;13) encoded PAX3/FKHR chimeric message, we have performed RT-PCR analysis on samples from 44 primary pediatric sarcomas and 8 sarcoma cell lines. PAX3/FKHR chimeric messages were detected in 24 of 27 alveolar, 2 of 12 embryonal, and 0 of 1 pleomorphic rhabdomyosarcoma and in 1 of 2 ectomesenchymomas. In contrast, none of 8 Ewing's sarcomas or 2 undifferentiated sarcomas expressed this message. Chimeric transcripts were detected in all cases with cytogenetic evidence of the (2;13) translocation, and in each case the chimeric PAX3/FKHR message had the identical junction sequence, suggesting that genomic chromosome breaks were clustered in a single intron in both genes. By combining the PAX3/FKHR RT-PCR assay with primers for detection of the Ewing's sarcoma t(11;22) encoded EWS/FLI-1 chimeric transcript, we have developed a multiplex RT-PCR reaction that allows the rapid and accurate identification of either translocation in a biopsy sample. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7887445

  1. Rapid detection and identification of human adenovirus species by adenoplex, a multiplex PCR-enzyme hybridization assay.

    PubMed

    Pehler-Harrington, Karen; Khanna, Marilyn; Waters, Chris R; Henrickson, Kelly J

    2004-09-01

    Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10(-2), 10(-1), 10(-1), 10(-2), 10(-1), and 10(-2), respectively. Similarly, the LOD for the six DNA controls ranged from 10(2) to 10(3) copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.

  2. Molecular characterization of Rhodococcus equi from horse-breeding farms by means of multiplex PCR for the vap gene family.

    PubMed

    Monego, Fernanda; Maboni, Franciele; Krewer, Cristina; Vargas, Agueda; Costa, Mateus; Loreto, Elgion

    2009-04-01

    This study evaluated the molecular characteristics of Rhodococcus equi isolates obtained from horses by a multiplex PCR assay that amplifies the vap gene family (vapA, -B, -C, -D, -E, -F, -G, and -H). A total of 180 R. equi isolates were studied from four different sources, namely healthy horse feces (112), soil (12), stalls (23), and clinical isolates (33) from horse-breeding farms. The technique was performed and confirmed by sequencing of amplified vap gene family controls. Thirty-two (17.8%) of the R. equi isolates were positive for the vapA gene and carried at least three other vap genes. All 147 isolates from equine feces, stalls, and soil failed to demonstrate any genes associated with virulence-inducing proteins. About 32 (97.0%) out of the 33 clinical equine isolates tested positive for the multiplex PCR assay for the vap gene family. They demonstrated six molecular profiles: 100% featured the vapA, vapD, and vapG genes, 86.6% vapF, 76.6% vapH, 43.3% vapC, 36.6% vapE, and none vapB. The most frequent molecular profile was vap A, -D, -F, G, and -H, where this profile was present in 37.5% of the strains. Moreover, there was no molecular epidemiological pattern for R. equi isolates that uniquely mapped to each horse-breeding farm studied. Our proposed technique allows the identification of eight members of the vap gene family (vapA, B, -C, -D, -E, -F, -G, and -H). It is a practical and efficient method of conducting clinical and epidemiological studies on R. equi isolates.

  3. Detection of HPV and co-infecting pathogens in healthy Italian women by multiplex real-time PCR.

    PubMed

    Camporiondo, Maria Pia; Farchi, Francesca; Ciccozzi, Massimo; Denaro, Aurelia; Gallone, Domenica; Maracchioni, Fabio; Favalli, Cartesio; Ciotti, Marco

    2016-01-01

    Several pathogens can be transmitted sexually and are an important cause of morbidity among sexually active women. The aim of the study was to detect the presence of human papillomavirus (HPV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP) in a group of 309 healthy women enrolled at the San Camillo - Forlanini hospital of Rome by using two multiplex real-time PCR assays based on TOCE® technology. The women's ages ranged from 34 to 60 years, median 49 [IQR 45-54]. Of the 309 women tested, HPV DNA was detected in 77/309 (24.9%) patients. Of these, 44 (14.2%) harboured a single infection while 33 (10.7%) were infected by multiple genotypes. Prevalence of HPV infection was highest among females aged 40-50 years (15.2%). Of the other pathogens sought, CT, MG and NG were not detected while positive results were found for MH (12/309, 3.9%), TV (4/309, 1.3%), UP (89/309, 28.8%) and UU (14/309, 4.5%). Co-infections were as follows: 5 MH/HPV, 4 TV/HPV, 34 UP/HPV and 9 UU/HPV. In HPV-positive women, the probability of being infected by UP and UU was 2.5 (p=0.00045) and 6 fold higher (p=0.0016) than in HPV-negative women. The study supports the use of multiplex real-time PCR assays in a routine diagnostic setting. The high sensitivity and specificity of these assays along with the simultaneous detection of the most common sexually transmitted pathogens confers an advantage with respect to more obsolete methods reducing costs and time to diagnosis.

  4. Detection of HPV and co-infecting pathogens in healthy Italian women by multiplex real-time PCR.

    PubMed

    Camporiondo, Maria Pia; Farchi, Francesca; Ciccozzi, Massimo; Denaro, Aurelia; Gallone, Domenica; Maracchioni, Fabio; Favalli, Cartesio; Ciotti, Marco

    2016-01-01

    Several pathogens can be transmitted sexually and are an important cause of morbidity among sexually active women. The aim of the study was to detect the presence of human papillomavirus (HPV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP) in a group of 309 healthy women enrolled at the San Camillo - Forlanini hospital of Rome by using two multiplex real-time PCR assays based on TOCE® technology. The women's ages ranged from 34 to 60 years, median 49 [IQR 45-54]. Of the 309 women tested, HPV DNA was detected in 77/309 (24.9%) patients. Of these, 44 (14.2%) harboured a single infection while 33 (10.7%) were infected by multiple genotypes. Prevalence of HPV infection was highest among females aged 40-50 years (15.2%). Of the other pathogens sought, CT, MG and NG were not detected while positive results were found for MH (12/309, 3.9%), TV (4/309, 1.3%), UP (89/309, 28.8%) and UU (14/309, 4.5%). Co-infections were as follows: 5 MH/HPV, 4 TV/HPV, 34 UP/HPV and 9 UU/HPV. In HPV-positive women, the probability of being infected by UP and UU was 2.5 (p=0.00045) and 6 fold higher (p=0.0016) than in HPV-negative women. The study supports the use of multiplex real-time PCR assays in a routine diagnostic setting. The high sensitivity and specificity of these assays along with the simultaneous detection of the most common sexually transmitted pathogens confers an advantage with respect to more obsolete methods reducing costs and time to diagnosis. PMID:27031891

  5. Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

    PubMed Central

    Kaewkong, Worasak; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Laummaunwai, Porntip; Lulitanond, Viraphong; Doanh, Pham Ngoc

    2013-01-01

    Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens. PMID:24516275

  6. Event-specific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Jia; Zhu, Shuifang; Miao, Haizhen; Huang, Wensheng; Qiu, Minyan; Huang, Yan; Fu, Xuping; Li, Yao

    2007-07-11

    With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes. PMID:17559227

  7. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

    PubMed

    Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

    2016-06-20

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  8. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

    PubMed Central

    Ståhlberg, Anders; Krzyzanowski, Paul M.; Jackson, Jennifer B.; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E.

    2016-01-01

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  9. Event-specific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Jia; Zhu, Shuifang; Miao, Haizhen; Huang, Wensheng; Qiu, Minyan; Huang, Yan; Fu, Xuping; Li, Yao

    2007-07-11

    With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes.

  10. Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in clinical isolates by multiplex PCR.

    PubMed

    Nakanaga, Kazue; Sekizuka, Tsuyoshi; Fukano, Hanako; Sakakibara, Yumi; Takeuchi, Fumihiko; Wada, Shinpei; Ishii, Norihisa; Makino, Masahiko; Kuroda, Makoto; Hoshino, Yoshihiko

    2014-01-01

    The rapidly growing mycobacterium M. abscessus sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures. M. abscessus sensu lato can be divided into at least three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Clinical isolates of rapidly growing mycobacteria were previously identified as M. abscessus by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as M. abscessus subsp. massiliense using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish M. abscessus subsp. massiliense from M. abscessus subsp. abscessus, a multiplex PCR assay, was developed using the whole-genome sequence of M. abscessus subsp. massiliense JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of M. abscessus subsp. massiliense from samples previously identified as M. abscessus. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays. PMID:24197885

  11. Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

    PubMed Central

    Li, Jiandong; Qu, Jing; He, Chengcheng; Zhang, Shuo; Li, Chuan; Zhang, Quanfu; Liang, Mifang; Li, Dexin

    2014-01-01

    Background Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Results Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Conclusions Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive

  12. Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in Clinical Stool Samples ▿

    PubMed Central

    Stark, D.; Al-Qassab, S. E.; Barratt, J. L. N.; Stanley, K.; Roberts, T.; Marriott, D.; Harkness, J.; Ellis, J. T.

    2011-01-01

    The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites. PMID:21048004

  13. Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

    PubMed

    Noorani, Md Salik; Awasthi, Prachi; Sharma, Maheshwar Prasad; Ram, Raja; Zaidi, Aijaz Asgar; Hallan, Vipin

    2013-10-01

    A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.

  14. Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

    PubMed Central

    Llewellyn, Stacey; Inpankaew, Tawin; Nery, Susana Vaz; Gray, Darren J.; Verweij, Jaco J.; Clements, Archie C. A.; Gomes, Santina J.; Traub, Rebecca; McCarthy, James S.

    2016-01-01

    Background Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy. Methodology/Principal Findings Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections. Conclusions/Significance Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and

  15. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

    PubMed Central

    Lamien, Charles Euloge; Spergser, Joachim; Lelenta, Mamadou; Wade, Abel; Gelaye, Esayas; Loitsch, Angelika; Minoungou, Germaine; Thiaucourt, Francois; Diallo, Adama

    2016-01-01

    Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples

  16. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.

    PubMed

    Settypalli, Tirumala Bharani Kumar; Lamien, Charles Euloge; Spergser, Joachim; Lelenta, Mamadou; Wade, Abel; Gelaye, Esayas; Loitsch, Angelika; Minoungou, Germaine; Thiaucourt, Francois; Diallo, Adama

    2016-01-01

    Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples

  17. Detection of enterotoxigenic Staphylococcus aureus in raw milk and dairy products by multiplex PCR.

    PubMed

    Gücükoğlu, Ali; Kevenk, T Onur; Uyanik, Tolga; Cadirci, Ozgür; Terzi, Göknur; Alişarli, Mustafa

    2012-11-01

    The aim of this study was to investigate the prevalence of enterotoxigenic Staphylococcus aureus in 122 samples, including 60 raw milk, 32 white cheese, 10 kashar cheese, 10 butter, and 10 ice cream samples obtained from Samsun province, Turkey. In this study, S. aureus was detected in 64 samples, including raw milk (45/60; 75%), white cheese (12/32; 37.5%), kashar cheese (3/10; 30%), butter (3/10; 30%), and ice cream (1/10; 10%) samples. A total of 81 isolates were identified as S. aureus by PCR with the presence of 16S rRNA and nuc genes. The presence of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, and SED was detected by multiplex PCR. According to the analysis, seven isolates from the raw milk samples (7/51; 13.7%) were enterotoxigenic; five of them produced SEA (5/7; 71.4%), one produced SEB (1/7; 14.2%), and one produced SEA+SEB (1/7; 14.2%). Four isolates from the white cheese samples (4/21; 19%) produced the SEA (1/4; 25%), SEC (1/4; 25%), SED (1/4; 25%), and SEA+SED (1/4; 25%) toxins. Two isolates from the kashar cheese samples (2/4; 50%) were found to be enterotoxigenic; one produced SEA (1/2; 50%) and the other produced SED (1/2; 50%). One isolate from the butter samples (1/4; 25%) showed enterotoxigenic character (SEB, 1/1; 100%). The products were found to be potentially hazardous to public health because of the fact that levels of contamination were higher than 105-106 cfu/g ml in 39% (25/64, 17 raw milk, 7 white cheese, and 1 butter) of the analyzed samples.

  18. Detection of enteroviruses and parechoviruses by a multiplex real-time RT-PCR assay.

    PubMed

    Pabbaraju, Kanti; Wong, Sallene; Wong, Anita A; Tellier, Raymond

    2015-04-01

    Detection of all enteroviruses while excluding cross-detection of rhinoviruses is challenging because of sequence similarities in the commonly used conserved targets for molecular assays. In addition, simultaneous detection and differentiation of enteroviruses and parechoviruses would be beneficial because of a similar clinical picture presented by these viruses. A sensitive and specific real-time RT-PCR protocol that can address these clinical needs would be valuable to molecular diagnostic laboratories. Here we report a multiplex nucleic acid based assay using hydrolysis probes targeting the 5' non-translated region for the detection and differentiation of enteroviruses and parechoviruses without cross-detection of rhinoviruses. This assay has been shown to detect enteroviruses belonging to the different species in a variety of specimen types without detecting the different species of rhinoviruses. Laboratory validation shows the assay to be sensitive, specific, reproducible, easy to set up and uses generic cycling conditions. This assay can be implemented for diagnostic testing of patient samples in a high throughput fashion.

  19. A multiplex real-time PCR assay for identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in samples from AIDS patients with opportunistic pneumonia.

    PubMed

    Gago, Sara; Esteban, Cristina; Valero, Clara; Zaragoza, Oscar; Puig de la Bellacasa, Jorge; Buitrago, María José

    2014-04-01

    A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r(2) of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

  20. Multiplex real-time RT-PCR for the simultaneous detection and quantification of GI, GII and GIV noroviruses.

    PubMed

    Farkas, Tibor; Singh, Amy; Le Guyader, Françoise S; La Rosa, Giuseppina; Saif, Linda; McNeal, Monica

    2015-10-01

    Noroviruses are important causes of acute gastroenteritis and are classified into six genogroups with GI, GII and GIV containing human pathogens. This high genetic diversity represents a significant challenge for diagnostic assay development. Genogroup specific monoplex and multiplex real time RT-PCR assays are widely used for the detection of GI and GII noroviruses. On the other hand, GIV norovirus detection is not part of routine laboratory diagnosis. This study describes the development and evaluation of a one tube, real time RT-PCR assay for the simultaneous detection and quantification of GI, GII and GIV noroviruses, including both GIV.1 (human) and GIV.2 (animal) strains. Assay performance was evaluated on a panel of norovirus positive clinical samples by comparison of monoplex and multiplex standard curves and Ct values. The multiplex assay demonstrated equal sensitivity and specificity to the monoplex assays and was able to detect all GI, GII and GIV noroviruses with Ct values equal to that of the monoplex assays. The multiplex assay described in this study will be instrumental for the better understanding of GIV norovirus epidemiology, including their possible zoonotic nature. PMID:26248055

  1. Simultaneous direct identification of genital microorganisms in voided urine using multiplex PCR-based reverse line blot assays.

    PubMed

    McKechnie, Michelle L; Kong, Fanrong; Gilbert, Gwendolyn L

    2013-01-01

    Our aim was to develop and evaluate sensitive methods that would allow simultaneous direct identification of multiple potential pathogens in clinical specimens for diagnosis and epidemiological studies, using a multiplex PCR-based reverse line blot assay. We have previously developed assays suitable for detection of bacterial respiratory and systemic pathogens. In this chapter we describe, in detail, a method developed to identify 14 genital microorganisms, for use in epidemiological studies of genital infection or colonization, using first voided urine specimens. The 14 urogenital pathogens or putative pathogens studied were Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, N. meningitidis, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Mycoplasma hominis, M. genitalium, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus 1 and 2, and adenovirus. Two species-specific primer pairs and probes were designed for each target. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism. In a clinical study among men attending sexual health clinics in Sydney, we used the assay to compare rates of detection of the 14 organisms in men with urethritis with those in asymptomatic controls and found the method to be sensitive, specific, convenient, and relatively inexpensive.

  2. Simultaneous direct identification of genital microorganisms in voided urine using multiplex PCR-based reverse line blot assays.

    PubMed

    McKechnie, Michelle L; Kong, Fanrong; Gilbert, Gwendolyn L

    2013-01-01

    Our aim was to develop and evaluate sensitive methods that would allow simultaneous direct identification of multiple potential pathogens in clinical specimens for diagnosis and epidemiological studies, using a multiplex PCR-based reverse line blot assay. We have previously developed assays suitable for detection of bacterial respiratory and systemic pathogens. In this chapter we describe, in detail, a method developed to identify 14 genital microorganisms, for use in epidemiological studies of genital infection or colonization, using first voided urine specimens. The 14 urogenital pathogens or putative pathogens studied were Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, N. meningitidis, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Mycoplasma hominis, M. genitalium, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus 1 and 2, and adenovirus. Two species-specific primer pairs and probes were designed for each target. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism. In a clinical study among men attending sexual health clinics in Sydney, we used the assay to compare rates of detection of the 14 organisms in men with urethritis with those in asymptomatic controls and found the method to be sensitive, specific, convenient, and relatively inexpensive. PMID:23104293

  3. Profound inhibition of the PCR step of CF V3 multiplex PCR/OLA assay by the use of UV-irradiated plastic reaction tubes.

    PubMed

    Fox, David H; Huang, Chih-Kang; Du, Juan; Chang, Tylis Y; Pan, Qiulu

    2007-06-01

    Supplies, such as bags of plastic reaction tubes, are sometimes left in the laminar flow hoods unintentionally while the ultraviolet (UV) lamp is illuminated overnight. In addition, UV irradiation is used for sterilization and amplicon inactivation to avoid contamination. The oligonucleotide ligation assay (OLA) is a unique approach to mutation detection of point mutations, small deletions, and small insertions. Recently, we encountered problems with this assay and peak heights were much lower or disappeared. After going through systemic trouble-shooting, we found that profound inhibition of the polymerase chain reaction (PCR) step of CF V3 multiplex PCR/OLA assay by the use of UV-irradiated plastic reaction tubes. When UV-irradiated tubes used throughout the assay, tubes exposed for 8 weeks at 0.7 m from the UV source gave a reduction of 60% and 67% in the assay products on the basis of sum of peak heights. Tubes exposed for 3 weeks at 0.1 m from the UV source totally eliminated assay product yielding no peaks. Further experiments showed that the inhibition happened mostly in the PCR step. Burgess and Hall had reported that inhibition of PCR of human glyceraldehydes-3-phosphate dehydrogenase transcripts after UV irradiating the tubes. This showed that the inhibition was not assay-specific. The reason that the inhibition of PCR was more profound could be due to a multiplex PCR assay and small reaction volume. The mechanism of PCR inhibition by UV irradiation is not clear. In conclusion, plastic reaction tubes intended for PCR/OLA assays should not be exposed to UV.

  4. Population data on 10 non-CODIS STR loci in Japanese population using a newly developed multiplex PCR system.

    PubMed

    Asamura, H; Ota, M; Fukushima, H

    2008-11-01

    This paper describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) systems for 10 loci (D1S1656, D2S1353, D8S1132, D12S1090, D14S608, D18S535, D19S253, D20S480, D21S226, and D22S689) unlinked to the core STR loci (non-CODIS loci). Of 252 samples taken from the Japanese population, PCR products ranged in length from 107 bp to 319 bp. No significant deviations from Hardy-Weinberg equilibrium were observed at any of the 10 loci. The accumulated power of discrimination and power of exclusion for the 10 loci were 0.999999999998 and 0.99991, respectively. We conclude that the present multiplex system for the 10 non-CODIS loci represents a powerful tool for forensic applications.

  5. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    PubMed

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.

  6. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    PubMed

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation. PMID:26410309

  7. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

    PubMed

    Simmons, Monika; Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  8. Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR.

    PubMed

    Zhang, Zhihong; Feng, Lixia; Xu, Hengyi; Liu, Chengwei; Shah, Nagendra P; Wei, Hua

    2016-02-01

    Bacillus cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe), and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus. The objective of this study was to develop a rapid and accurate method that combines multiplex PCR with propidium monoazide to selectively detect viable cells of enterotoxin-producing B. cereus in milk powder, noodles, and rice, and investigate the distribution of enterotoxins in 62 strains of B. cereus in Jiangxi province, China. The specificity of primers of 3 enterotoxins (i.e., cytK, nheA, and hblD) of B. cereus was verified by inclusivity and exclusivity tests using single PCR. Upon optimization of multiplex PCR conditions, it was found that the detection limit of viable cells was 10(2) cfu/mL of B. cereus in pure culture. By enrichment for 3 or 4 h and propidium monoazide pretreatment, a protocol for detection of viable cells as low as 2.2×10(1) cfu/g in spiked food (e.g., milk powder, noodles, and rice) was established and proved valid even under the interference of non-Bacillus cereus at as high as 10(5) cfu/g. Moreover, the protocol based on multiplex PCR for detection was applied for the analysis of distribution of toxin gene of B. cereus, and the results showed a regional feature for toxin gene distribution, indicating that potential toxigenicity of B. cereus should be evaluated further.

  9. A single-tube duplex and multiplex PCR for simultaneous detection of four cassava mosaic begomovirus species in cassava plants.

    PubMed

    Aloyce, R C; Tairo, F; Sseruwagi, P; Rey, M E C; Ndunguru, J

    2013-04-01

    A single-tube duplex and multiplex PCR was developed for the simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava mosaic Zanzibar virus (EACMZV), four cassava mosaic begomoviruses (CMBs) affecting cassava in sub-Saharan Africa. Co-occurrence of the CMBs in cassava synergistically enhances disease symptoms and complicates their detection and diagnostics. Four primer pairs were designed to target DNA-A component sequences of cassava begomoviruses in a single tube PCR amplification using DNA extracted from dry-stored cassava leaves. Duplex and multiplex PCR enabled the simultaneous detection and differentiation of the four CMBs, namely ACMV (940bp), EACMCV (435bp), EACMMV (504bp) and EACMZV (260bp) in single and mixed infections, and sequencing results confirmed virus identities according to the respective published sequences of begomovirus species. In addition, we report here a modified Dellapotra et al. (1983) protocol, which was used to extract DNA from dry and fresh cassava leaves with comparable results. Using the duplex and multiplex techniques, time was saved and amount of reagents used were reduced, which translated into reduced cost of the diagnostics. This tool can be used by cassava breeders screening for disease resistance; scientists doing virus diagnostic studies; phytosanitary officers checking movement of diseased planting materials, and seed certification and multipliers for virus indexing.

  10. Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients

    PubMed Central

    Moltzahn, Felix; Olshen, Adam B.; Baehner, Lauren; Peek, Andrew; Fong, Lawrence; Stöppler, Hubert; Simko, Jeffry; Hilton, Joan F.; Carroll, Peter; Blelloch, Robert

    2010-01-01

    Recent prostate specific antigen (PSA) based screening trials indicate an urgent need for novel and non-invasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Non-coding microRNAs (miRNAs) in the serum and plasma have been shown to have potential as non-invasive markers for physiological and pathological conditions. To identify serum miRNAs that diagnose and correlate with prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR (qRT-PCR) method involving purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA - deficient) mouse ES cells (mESC) as the benchmark, we confirmed the validity of our technique, while uncovering a significant lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA (Cancer of the Prostate Risk Assessment) scores, we identified miRNA signatures that allow to diagnose cancer patients and correlate with prognosis. These serum signatures include oncogenic and tumor suppressive miRNAs suggesting functional roles in prostate cancer progression. PMID:21098088

  11. Development of a single tube multiplex real-time PCR to detect the most clinically relevant Mucormycetes species.

    PubMed

    Bernal-Martínez, L; Buitrago, M J; Castelli, M V; Rodriguez-Tudela, J L; Cuenca-Estrella, M

    2013-01-01

    Mucormycetes infections are very difficult to treat and a delay in diagnosis could be fatal for the outcome of the patient. A molecular diagnostic technique based on Real Time PCR was developed for the simultaneous detection of Rhizopus oryzae, Rhizopus microsporus and the genus Mucor spp. in both culture and clinical samples. The methodology used was Molecular beacon species-specific probes with an internal control. This multiplex real-time PCR (MRT-PCR) was tested in 22 cultured strains and 12 clinical samples from patients suffering from a proven mucormycosis. Results showed 100% specificity and a detection limit of 1 fg of DNA per microlitre of sample. The sensitivity was 100% for clinical cultured strains and for clinical samples containing species detected by the PCR assay. Other mucormycetes species were not detected in clinical samples. This technique can be useful for clinical diagnosis and further studies are warranted.

  12. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

    PubMed Central

    2012-01-01

    Background Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD. Results A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT). Conclusions The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD. PMID:22455597

  13. Development of a novel hexa-plex PCR method for identification and serotyping of Salmonella species.

    PubMed

    Li, Ruichao; Wang, Yang; Shen, Jianzhong; Wu, Congming

    2014-01-01

    Salmonella is one of the most important foodborne pathogens, which causes a huge economic burden worldwide. To detect Salmonella rapidly is very meaningful in preventing salmonellosis and decreasing economic losses. Currently, isolation of Salmonella is confirmed by biochemical and serobased serotyping methods, which are time consuming, labor intensive, and complicated. To solve this problem, a hexa-plex polymerase chain reaction (PCR) method was developed using comparative genomics analysis and multiplex PCR technology to detect Salmonella and Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Agona, Salmonella Choleraesuis, and Salmonella Pullorum simultaneously. The accuracy of this method was tested by a collection of 142 Salmonella. Furthermore, the strategy described in this article to mine serovar-specific fragments for Salmonella could be used to find specific fragments for other Salmonella serotypes and bacteria. The combination of this strategy and multiplex PCR is promising in the rapid identification of foodborne pathogens.

  14. Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing

    PubMed Central

    Schubert, Claudia; Chatain, Nicolas; Sontag, Stephanie; Isfort, Susanne; Ortiz-Brüchle, Nadina; Schmitt, Karla; Krüger, Luisa; Zerres, Klaus; Zenke, Martin; Brümmendorf, Tim H.; Koschmieder, Steffen

    2015-01-01

    In order to assess the feasibility of amplicon-based parallel next generation sequencing (NGS) for the diagnosis of myeloproliferative neoplasms (MPN), we investigated multiplex-PCR of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes. Samples from 64 patients with MPN and five controls as well as seven (myeloid) cell lines were analyzed. Healthy donor and reactive erythrocytosis samples showed several frequent single-nucleotide polymorphisms (SNPs) but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of the known mutations. In the patient samples, JAK2 V617F was present in all PV, 4 of 10 ET, and 16 of 19 MF patients. The JAK2 V617F allele burden was different in the three groups (ET, 33+/-22%; PV 48+/-28% and MF 68+/- 29%). Further analysis detected both previously described and undescribed mutations (i.e., G12V NRAS, IDH1 R132H, E255G ABL, and V125G IDH1 mutations). One patient with lymphoid BC/Ph+ ALL who harbored a T315I ABL mutation and was treated with ponatinib was found to have developed a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation might have caused ponatinib resistance in this patient. In conclusion, amplicon-sequencing-based NGS allows simultaneous analysis of multiple MPN associated genes for diagnosis and during treatment and measurement of the mutant allele burden. PMID:25894969

  15. Rapid detection of hepatitis B virus variants associated with lamivudine and adefovir resistance by multiplex ligation-dependent probe amplification combined with real-time PCR.

    PubMed

    Jia, Shuangrong; Wang, Feng; Li, Fake; Chang, Kai; Yang, Shaojun; Zhang, Kejun; Jiang, Wenbin; Shang, Ya; Deng, Shaoli; Chen, Ming

    2014-02-01

    Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.

  16. Rapid Detection of Hepatitis B Virus Variants Associated with Lamivudine and Adefovir Resistance by Multiplex Ligation-Dependent Probe Amplification Combined with Real-Time PCR

    PubMed Central

    Jia, Shuangrong; Wang, Feng; Li, Fake; Chang, Kai; Yang, Shaojun; Zhang, Kejun; Jiang, Wenbin; Shang, Ya

    2014-01-01

    Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice. PMID:24478474

  17. Simultaneous Detection of Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Salmonella spp., and Staphylococcus aureus in Low-fatted Milk by Multiplex PCR

    PubMed Central

    Kim, Ji-Hyun; Rhim, Seong-Ryul; Kim, Kee-Tae; Paik, Hyun-Dong

    2014-01-01

    A rapid and specific PCR assay for the simultaneous detection of Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Salmonella spp., and Staphylococcus aureus in foods was developed to reduce the detection time and to increase sensitivity. Multiplex PCR developed in this study produced only actA, fliC, hbl, invA, ileS amplicons, but did not produce any non-specific amplicon. The primer sets successfully amplified the target genes in the multiplex PCR without any non-specific or additional bands on the other strains. The multiplex PCR assays also amplified some target genes from five pathogens, and multiplex amplification was obtained from as little as 1 pg of DNA. According to the results from the sensitivity evaluation, the multiplex PCR developed in this study detected 10 cells/mL of the pathogens inoculated in milk samples, respectively. The results suggested that multiplex PCR was an effective assay demonstrating high specificity for the simultaneous detection of five target pathogens in food system. PMID:26761507

  18. The current incidence of viral disease in korean sweet potatoes and development of multiplex rt-PCR assays for simultaneous detection of eight sweet potato viruses.

    PubMed

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-12-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

  19. The current incidence of viral disease in korean sweet potatoes and development of multiplex rt-PCR assays for simultaneous detection of eight sweet potato viruses.

    PubMed

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-12-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded. PMID:25506306

  20. The Current Incidence of Viral Disease in Korean Sweet Potatoes and Development of Multiplex RT-PCR Assays for Simultaneous Detection of Eight Sweet Potato Viruses

    PubMed Central

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-01-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded. PMID:25506306

  1. Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

    PubMed

    Wang, Ming; Escudero-Ibarz, Leire; Moody, Sarah; Zeng, Naiyan; Clipson, Alexandra; Huang, Yuanxue; Xue, Xuemin; Grigoropoulos, Nicholas F; Barrans, Sharon; Worrillow, Lisa; Forshew, Tim; Su, Jing; Firth, Andrew; Martin, Howard; Jack, Andrew; Brugger, Kim; Du, Ming-Qing

    2015-09-01

    High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues.

  2. Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

    PubMed Central

    Wang, Ming; Escudero-Ibarz, Leire; Moody, Sarah; Zeng, Naiyan; Clipson, Alexandra; Huang, Yuanxue; Xue, Xuemin; Grigoropoulos, Nicholas F.; Barrans, Sharon; Worrillow, Lisa; Forshew, Tim; Su, Jing; Firth, Andrew; Martin, Howard; Jack, Andrew; Brugger, Kim; Du, Ming-Qing

    2016-01-01

    High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues. PMID:26165823

  3. Identification of viable Listeria species based on reverse transcription-multiplex PCR (RT-MPCR) and restriction digestion.

    PubMed

    Rattanachaikunsopon, Pongsak; Phumkhachorn, Parichat

    2012-01-01

    A novel method for the identification of viable Listeria species was developed based on reverse transcription-multiplex PCR (RT-MPCR) and restriction digestion. The targets for RT-MPCR were iap mRNAs whose genes are common to all Liseria species. A set of five primers was used in this study. Two of them were genus specific, and the other three were specific to L. monocytogenase, L. innocua, and L. grayi respectively. By RT-MPCR, L. monocytogenese, L. innocua, L. grayi, and a group of Listeria species, including L. ivanovii, L. welshimeri, and L. seeligeri, were specifically identified. To differentiate the latter three Listeria species, RT-MPCR products were subjected to digestion with HpaI and ScaI. The sensitivity of RT-MPCR in detecting Listeria species was determined to be 50 CFU/mL. RT-MPCR was found to discriminate between viable and nonviable cells and to detect viable Listeria species in a food model.

  4. Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

    PubMed

    Wang, Ming; Escudero-Ibarz, Leire; Moody, Sarah; Zeng, Naiyan; Clipson, Alexandra; Huang, Yuanxue; Xue, Xuemin; Grigoropoulos, Nicholas F; Barrans, Sharon; Worrillow, Lisa; Forshew, Tim; Su, Jing; Firth, Andrew; Martin, Howard; Jack, Andrew; Brugger, Kim; Du, Ming-Qing

    2015-09-01

    High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues. PMID:26165823

  5. Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group.

    PubMed

    Yang, I-Chen; Shih, Daniel Yang-Chih; Huang, Tsui-Ping; Huang, Yun-Pu; Wang, Jan-Yi; Pan, Tzu-Ming

    2005-10-01

    Five different enterotoxins and one emetic toxin of Bacillus cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex PCR with 12 primer pairs was established. In developing the assay method, a common terminal sequence at the 3' ends of all primers was chosen and a hot start Taq polymerase was used to overcome primer dimer formation. The assay was successfully applied to analyze the toxigenic potential of 162 food-poisoning and food-related strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of Bacillus mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains (P < 0.05), although the two species were closely related. The results suggest that many B. mycoides strains might be less prone to cause food poisoning. They also indicate the importance of detecting the toxin genes together with the detection of the species in the B. cereus group.

  6. Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

    PubMed

    Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

    2003-12-15

    Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

  7. Dynamic multiplexed analysis method using ion mobility spectrometer

    SciTech Connect

    Belov, Mikhail E

    2010-05-18

    A method for multiplexed analysis using ion mobility spectrometer in which the effectiveness and efficiency of the multiplexed method is optimized by automatically adjusting rates of passage of analyte materials through an IMS drift tube during operation of the system. This automatic adjustment is performed by the IMS instrument itself after determining the appropriate levels of adjustment according to the method of the present invention. In one example, the adjustment of the rates of passage for these materials is determined by quantifying the total number of analyte molecules delivered to the ion trap in a preselected period of time, comparing this number to the charge capacity of the ion trap, selecting a gate opening sequence; and implementing the selected gate opening sequence to obtain a preselected rate of analytes within said IMS drift tube.

  8. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

    PubMed Central

    Chung, Yousun; Kim, Taek Soo; Min, Young Gi; Hong, Yun Ji; Park, Jeong Su; Hwang, Sang Mee; Song, Kyoung-Ho; Kim, Eu Suk; Kim, Hong Bin; Song, Junghan; Kim, Eui-Chong

    2016-01-01

    Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. epidermidis (MRSE), methicillin-susceptible S. epidermidis (MSSE), methicillin-resistant non-S. epidermidis CoNS (MRCoNS), and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS) (κ = 0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings. PMID:27403436

  9. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia.

    PubMed

    Chung, Yousun; Kim, Taek Soo; Min, Young Gi; Hong, Yun Ji; Park, Jeong Su; Hwang, Sang Mee; Song, Kyoung-Ho; Kim, Eu Suk; Park, Kyoung Un; Kim, Hong Bin; Song, Junghan; Kim, Eui-Chong

    2016-01-01

    Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. epidermidis (MRSE), methicillin-susceptible S. epidermidis (MSSE), methicillin-resistant non-S. epidermidis CoNS (MRCoNS), and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS) (κ = 0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings. PMID:27403436

  10. A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience.

    PubMed

    Nielsen, Alex Christian Yde; Böttiger, Blenda; Midgley, Sofie Elisabeth; Nielsen, Lars Peter

    2013-11-01

    As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay. The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses in 171 (8%) and 66 (3%) of the samples, respectively. 180 of the positive samples could be genotyped by PCR and sequencing and the most common genotypes found were human parechovirus type 3, echovirus 9, enterovirus 71, Coxsackievirus A16, and echovirus 25. During 2009 in Denmark, both enterovirus and human parechovirus type 3 had a similar seasonal pattern with a peak during the summer and autumn. Human parechovirus type 3 was almost invariably found in children less than 4 months of age. In conclusion, a multiplex assay was developed allowing simultaneous detection of 2 viruses, which can cause similar clinical symptoms.

  11. DNA analysis using an integrated microchip for multiplex PCR amplification and electrophoresis for reference samples.

    PubMed

    Le Roux, Delphine; Root, Brian E; Reedy, Carmen R; Hickey, Jeffrey A; Scott, Orion N; Bienvenue, Joan M; Landers, James P; Chassagne, Luc; de Mazancourt, Philippe

    2014-08-19

    A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.

  12. Digital gene expression analysis with sample multiplexing and PCR duplicate detection: A straightforward protocol.

    PubMed

    Rozenberg, Andrey; Leese, Florian; Weiss, Linda C; Tollrian, Ralph

    2016-01-01

    Tag-Seq is a high-throughput approach used for discovering SNPs and characterizing gene expression. In comparison to RNA-Seq, Tag-Seq eases data processing and allows detection of rare mRNA species using only one tag per transcript molecule. However, reduced library complexity raises the issue of PCR duplicates, which distort gene expression levels. Here we present a novel Tag-Seq protocol that uses the least biased methods for RNA library preparation combined with a novel approach for joint PCR template and sample labeling. In our protocol, input RNA is fragmented by hydrolysis, and poly(A)-bearing RNAs are selected and directly ligated to mixed DNA-RNA P5 adapters. The P5 adapters contain i5 barcodes composed of sample-specific (moderately) degenerate base regions (mDBRs), which later allow detection of PCR duplicates. The P7 adapter is attached via reverse transcription with individual i7 barcodes added during the amplification step. The resulting libraries can be sequenced on an Illumina sequencer. After sample demultiplexing and PCR duplicate removal with a free software tool we designed, the data are ready for downstream analysis. Our protocol was tested on RNA samples from predator-induced and control Daphnia microcrustaceans. PMID:27401671

  13. Methods for threshold determination in multiplexed assays

    SciTech Connect

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2014-06-24

    Methods for determination of threshold values of signatures comprised in an assay are described. Each signature enables detection of a target. The methods determine a probability density function of negative samples and a corresponding false positive rate curve. A false positive criterion is established and a threshold for that signature is determined as a point at which the false positive rate curve intersects the false positive criterion. A method for quantitative analysis and interpretation of assay results together with a method for determination of a desired limit of detection of a signature in an assay are also described.

  14. Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces.

    PubMed

    Shridhar, P B; Noll, L W; Shi, X; An, B; Cernicchiaro, N; Renter, D G; Nagaraja, T G; Bai, J

    2016-01-01

    Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture-spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P > 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

  15. Identification of markers tightly linked to tomato yellow leaf curl disease and root-knot nematode resistance by multiplex PCR.

    PubMed

    Chen, S X; Du, J N; Hao, L N; Wang, C Y; Chen, Q; Chang, Y X

    2012-01-01

    Seven different commercial F₁ hybrids and two F₂ populations were evaluated by multiplex PCR to identify plants that are homozygous or heterozygous for Ty-1 and Mi, which confer resistance to tomato yellow leaf curl disease and root-knot nematode, respectively. The Ty-1 and Mi markers were amplified by PCR and identified by digestion of the amplicons with the TaqI enzyme. The hybrids E13 and 288 were found to be Ty/ty heterozygous plants with 398-, 303-, and 95-bp bands, and B08, 314, 198, and A10 were found to be ty/ty homozygous plants with a 398-bp band; whereas 098 did not give any PCR products. The hybrids E13 and 198 were found to be Mi/Mi homozygous plants with 570- and 180-bp bands, and 288 and A10 were found to be Mi/mi heterozygous plants, with 750-, 570- and 180-bp bands, and B08, 109 and 314 were found to be mi/mi homozygous plants with only a 750-bp band. We additionally developed a multiplex PCR technique for JB-1 and Mi, which confer resistance to tomato yellow leaf curl disease and root-knot nematode. The JB-1 marker identified the genotype of the Ty gene, and the plants that produced the 400-bp band were ty/ty homozygous plants, whereas the plants that produced 400- and 500-bp bands were resistant to tomato yellow leaf curl disease. We conclude that multiplex PCRs can be used to reproducibly and efficiently detect these resistance genes. PMID:22869069

  16. Development of a Multiplex-PCR assay for the rapid identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus.

    PubMed

    Pennacchia, Carmela; Breeuwer, Pieter; Meyer, Rolf

    2014-10-01

    The presence of thermophilic bacilli in dairy products is indicator of poor hygiene. Their rapid detection and identification is fundamental to improve the industrial reactivity in the implementation of corrective and preventive actions. In this study a rapid and reliable identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus was achieved by species-specific PCR assays. Two primer sets, targeting the ITS 16S-23S rRNA region and the rpoB gene sequence of the target species respectively, were employed. Species-specificity of both primer sets was evaluated by using 53 reference strains of DSMZ collection; among them, 13 species of the genus Geobacillus and 15 of the genus Anoxybacillus were represented. Moreover, 99 wild strains and 23 bulk cells collected from 24 infant formula powders gathered from several countries worldwide were included in the analyses. Both primer sets were highly specific and the expected PCR fragments were obtained only when DNA from G. stearothermophilus or A. flavithermus was used. After testing their specificity, they were combined in a Multiplex-PCR assay for the simultaneous identification of the two target species. The specificity of the Multiplex-PCR was evaluated by using both wild strains and bulk cells. Every analysis confirmed the reliable identification results provided by the single species-specific PCR methodology. The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the PCR procedures developed in this study highlight the advantage of their application for the specific detection and identification of the thermophilic species G. stearothermophilus and A. flavithermus. PMID:24929881

  17. Development of a Multiplex-PCR assay for the rapid identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus.

    PubMed

    Pennacchia, Carmela; Breeuwer, Pieter; Meyer, Rolf

    2014-10-01

    The presence of thermophilic bacilli in dairy products is indicator of poor hygiene. Their rapid detection and identification is fundamental to improve the industrial reactivity in the implementation of corrective and preventive actions. In this study a rapid and reliable identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus was achieved by species-specific PCR assays. Two primer sets, targeting the ITS 16S-23S rRNA region and the rpoB gene sequence of the target species respectively, were employed. Species-specificity of both primer sets was evaluated by using 53 reference strains of DSMZ collection; among them, 13 species of the genus Geobacillus and 15 of the genus Anoxybacillus were represented. Moreover, 99 wild strains and 23 bulk cells collected from 24 infant formula powders gathered from several countries worldwide were included in the analyses. Both primer sets were highly specific and the expected PCR fragments were obtained only when DNA from G. stearothermophilus or A. flavithermus was used. After testing their specificity, they were combined in a Multiplex-PCR assay for the simultaneous identification of the two target species. The specificity of the Multiplex-PCR was evaluated by using both wild strains and bulk cells. Every analysis confirmed the reliable identification results provided by the single species-specific PCR methodology. The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the PCR procedures developed in this study highlight the advantage of their application for the specific detection and identification of the thermophilic species G. stearothermophilus and A. flavithermus.

  18. Data transformation methods for multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  19. Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei

    PubMed Central

    2013-01-01

    Background Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Methods Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. Results A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. Conclusions The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification. PMID:23409683

  20. Direct-multiplex PCR assay for meat species identification in food products.

    PubMed

    Kitpipit, Thitika; Sittichan, Kuangtiwa; Thanakiatkrai, Phuvadol

    2014-11-15

    This is the first time that direct PCR - DNA amplification without prior DNA extraction - was successfully developed and fully validated for rapid and economical simultaneous identification of six commonly consumed meat species. To achieve this, six species-specific primers were selected from previous reports and newly designed from the mitochondrial cytochrome b (cyt b), cytochrome oxidase I (COI), and 12s rRNA gene. The assay generated PCR products of 100, 119, 133, 155, 253, and 311 bp for pork, lamb/mutton, chicken, ostrich meat, horsemeat and beef, respectively. Validation showed that the assay is robust, rapid, economical, reproducible, specific, and sensitive down to 12,500 mitochondrial copy (equating to seven fg). It could be used with a variety of raw meats and products, including highly degraded and processed food samples. This proposed method will be greatly beneficial to the consumers, food industry, and law enforcement.

  1. Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples.

    PubMed

    Chiang, Yu-Cheng; Tsen, Hau-Yang; Chen, Hsin-Yen; Chang, Yu-Hsin; Lin, Chien-Ku; Chen, Chih-Yuan; Pai, Wan-Yu

    2012-01-01

    Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10(0) target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis. PMID:22101309

  2. Application of multiplex PCR approaches for shark molecular identification: feasibility and applications for fisheries management and conservation in the Eastern Tropical Pacific.

    PubMed

    Caballero, S; Cardeñosa, D; Soler, G; Hyde, J

    2012-03-01

    Here we describe the application of new and existing multiplex PCR methodologies for shark species molecular identification. Four multiplex systems (group ID, thresher sharks, hammerhead sharks and miscellaneous shark) were employed with primers previously described and some designed in this study, which allow for species identification after running PCR products through an agarose gel. This system was implemented for samples (bodies and fins) collected from unidentified sharks landed in the port of Buenaventura and from confiscated tissues obtained from illegal fishing around the Malpelo Island Marine Protected Area, Pacific Coast of Colombia. This method has allowed reliable identification, to date, of 407 samples to the genus and/or species levels, most of them (380) identified as the pelagic thresher shark (Alopias pelagicus). Another seven samples were identified as scalloped hammerhead sharks (Sphyrna lewini). This is an easy-to-implement and reliable identification method that could even be used locally to monitor shark captures in the main fishing ports of developed and developing countries.

  3. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer.

    PubMed

    Vargas, Diana Y; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A E

    2016-01-01

    We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

  4. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

    PubMed Central

    Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.

    2016-01-01

    We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

  5. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer.

    PubMed

    Vargas, Diana Y; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A E

    2016-01-01

    We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.

  6. Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

    PubMed

    Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A

    2015-01-01

    Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

  7. Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

    PubMed Central

    2014-01-01

    Background Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. PMID:24997675

  8. Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia.

    PubMed

    Ben M'hadheb, Manel; Harrabi, Myriam; Souii, Amira; Jrad-Battikh, Nadia; Gharbi, Jawhar

    2015-03-01

    Influenza viruses are negative stranded segmented RNA viruses belonging to Orthomyxoviridae family. They are classified into three types A, B, and C. Type A influenza viruses are classified into subtypes according to the antigenic characters of the surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The aim of the present study is to develop a fast and reliable multiplex RT-PCR technique for detecting simultaneously the subtypes A/H1N1 and A/H3N2 of influenza virus. Our study included 398 patients (mean age 30.33 ± 19.92 years) with flu or flu-like syndromes, consulting physicians affiliated with collaborating teams. A multiplex RT-PCR detecting A/H1N1 and A/H3N2 influenza viruses and an examination by indirect immunofluorescence (IFI) were performed. In the optimized conditions, we diagnosed by IFI a viral infection in 90 patients (22.6 %): 85 cases of influenza type A, four cases of influenza type B, and only one case of coinfection with types A and B. An evaluation of the technique was performed on 19 clinical specimens positive in IFI, and we detected eight cases of A/H3N2, five cases of A/H1N1, one case of influenza virus type A which is not an H1N1 nor H3N2, and five negative cases. Multiplex RT-PCR is a sensitive technique allowing an effective and fast diagnosis of respiratory infections caused by influenza viruses in which the optimization often collides with problems of sensibility.

  9. Development of a High-Throughput Multiplex PCR and Capillary Electrophoresis Technique for Serotype Determination of Salmonella Enterica Food Animal Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Previously, a multiplex PCR technique was developed to identify the top 30 human clinical serotypes of Salmonella enterica. To improve the speed, ease of use, utility and discriminatory ability of the technique, additional primers were added and the PCR product discrimination and analysi...

  10. Multiplex real-time PCR using SYBR® GreenER™ for the detection of DNA allergens in food.

    PubMed

    Pafundo, Simona; Gullì, Mariolina; Marmiroli, Nelson

    2010-03-01

    We describe the development of a six-target real-time multiplex PCR assay with the SYBR® GreenER™ fluorescent dye, targeted to genes encoding for allergenic proteins commonly present in many processed food products (patent application pending). The assay was successfully trialled on reconstructed food matrices and on a range of commercial foodstuffs, and is proposed as a ready-to-use analytical tool for food manufacturers to detect the presence or confirm the absence of sequences encoding for important allergenic proteins of plant origin.

  11. Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries.

    PubMed

    Onuk, Ertan Emek; Ciftci, Alper; Findik, Arzu; Durmaz, Yuksel

    2010-09-01

    Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.

  12. Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

    PubMed Central

    Onuk, Ertan Emek; Findik, Arzu; Durmaz, Yuksel

    2010-01-01

    Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria. PMID:20706031

  13. [Simultaneous detection of respiratory viruses and influenza A virus subtypes using multiplex PCR].

    PubMed

    Ciçek, Candan; Bayram, Nuri; Anıl, Murat; Gülen, Figen; Pullukçu, Hüsnü; Saz, Eylem Ulaş; Telli, Canan; Cok, Gürsel

    2014-10-01

    This study was conducted to investigate the respiratory viruses and subtyping of influenza A virus when positive by multiplex PCR in patients with flu-like symptoms, after the pandemic caused by influenza A (H1N1)pdm09. Nasopharyngeal swab samples collected from 700 patients (313 female, 387 male; age range: 24 days-94 yrs, median age: 1 yr) between December 2010 - January 2013 with flu-like symptoms including fever, headache, sore throat, rhinitis, cough, myalgia as defined by the World Health Organization were included in the study. Nucleic acid extractions (Viral DNA/RNA Extraction Kit, iNtRON, South Korea) and cDNA synthesis (RevertAid First Strand cDNA Synthesis Kits, Fermentas, USA) were performed according to the manufacturer's protocol. Multiplex amplification of nucleic acids was performed using DPO (dual priming oligonucleotide) primers and RV5 ACE Screening Kit (Seegene, South Korea) in terms of the presence of influenza A (INF-A) virus, influenza B (INF-B) virus, respiratory syncytial virus (RSV), and the other respiratory viruses. PCR products were detected by automated polyacrylamide gel electrophoresis using Screen Tape multiple detection system. Specimens which were positive for viral nucleic acids have been further studied by using specific DPO primers, FluA ACE Subtyping and RV15 Screening (Seegene, South Korea) kits. Four INF-A virus subtypes [human H1 (hH1), human H3 (hH3), swine H1 (sH1), avian H5 (aH5)] and 11 other respiratory viruses [Adenovirus, parainfluenza virus (PIV) types 1-4, human bocavirus (HBoV), human metapneumovirus (HMPV), rhinovirus types A and B, human coronaviruses (HCoV) OC43, 229E/NL63] were investigated with those tests. In the study, 53.6% (375/700) of the patients were found to be infected with at least one virus and multiple respiratory virus infections were detected in 15.7% (59/375) of the positive cases, which were mostly (49/59, 83%) in pediatric patients. RSV and rhinovirus coinfections were the most prevalent (18

  14. Combining forces--the use of Landsat TM satellite imagery, soil parameter information, and multiplex PCR to detect Coccidioides immitis growth sites in Kern County, California.

    PubMed

    Lauer, Antje; Talamantes, Jorge; Castañón Olivares, Laura Rosío; Medina, Luis Jaime; Baal, Joe Daryl Hugo; Casimiro, Kayla; Shroff, Natasha; Emery, Kirt W

    2014-01-01

    Coccidioidomycosis is a fungal disease acquired through the inhalation of spores of Coccidioides spp., which afflicts primarily humans and other mammals. It is endemic to areas in the southwestern United States, including the San Joaquin Valley portion of Kern County, California, our region of interest (ROI). Recently, incidence of coccidioidomycosis, also known as valley fever, has increased significantly, and several factors including climate change have been suggested as possible drivers for this observation. Up to date details about the ecological niche of C. immitis have escaped full characterization. In our project, we chose a three-step approach to investigate this niche: 1) We examined Landsat-5-Thematic-Mapper multispectral images of our ROI by using training pixels at a 750 m × 750 m section of Sharktooth Hill, a site confirmed to be a C. immitis growth site, to implement a Maximum Likelihood Classification scheme to map out the locations that could be suitable to support the growth of the pathogen; 2) We used the websoilsurvey database of the US Department of Agriculture to obtain soil parameter data; and 3) We investigated soil samples from 23 sites around Bakersfield, California using a multiplex Polymerase Chain Reaction (PCR) based method to detect the pathogen. Our results indicated that a combination of satellite imagery, soil type information, and multiplex PCR are powerful tools to predict and identify growth sites of C. immitis. This approach can be used as a basis for systematic sampling and investigation of soils to detect Coccidioides spp.

  15. Combining Forces - The Use of Landsat TM Satellite Imagery, Soil Parameter Information, and Multiplex PCR to Detect Coccidioides immitis Growth Sites in Kern County, California

    PubMed Central

    Lauer, Antje; Talamantes, Jorge; Castañón Olivares, Laura Rosío; Medina, Luis Jaime; Baal, Joe Daryl Hugo; Casimiro, Kayla; Shroff, Natasha; Emery, Kirt W.

    2014-01-01

    Coccidioidomycosis is a fungal disease acquired through the inhalation of spores of Coccidioides spp., which afflicts primarily humans and other mammals. It is endemic to areas in the southwestern United States, including the San Joaquin Valley portion of Kern County, California, our region of interest (ROI). Recently, incidence of coccidioidomycosis, also known as valley fever, has increased significantly, and several factors including climate change have been suggested as possible drivers for this observation. Up to date details about the ecological niche of C. immitis have escaped full characterization. In our project, we chose a three-step approach to investigate this niche: 1) We examined Landsat-5-Thematic-Mapper multispectral images of our ROI by using training pixels at a 750 m×750 m section of Sharktooth Hill, a site confirmed to be a C. immitis growth site, to implement a Maximum Likelihood Classification scheme to map out the locations that could be suitable to support the growth of the pathogen; 2) We used the websoilsurvey database of the US Department of Agriculture to obtain soil parameter data; and 3) We investigated soil samples from 23 sites around Bakersfield, California using a multiplex Polymerase Chain Reaction (PCR) based method to detect the pathogen. Our results indicated that a combination of satellite imagery, soil type information, and multiplex PCR are powerful tools to predict and identify growth sites of C. immitis. This approach can be used as a basis for systematic sampling and investigation of soils to detect Coccidioides spp. PMID:25380290

  16. A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

    SciTech Connect

    Letant, S E; .Ortiz, J I; Tammero, L; Birch, J M; Derlet, R W; Cohen, S; Manning, D; McBride, M T

    2007-04-11

    We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. This article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.

  17. Development of a multiplex PCR for rapid detection of verocytotoxin-producing Escherichia coli O26 in raw milk and ground beef.

    PubMed

    Lorusso, V; Dambrosio, A; Quaglia, N C; Parisi, A; Lasalandra, G; Mula, G; Virgilio, S; Lucifora, G; Dario, M; Normanno, G

    2011-01-01

    Verocytotoxin-producing Escherichia coli (VTEC) O26 is an emergent pathotype that has caused an increasing number of sporadic cases and outbreaks of gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome in the United States and Europe. Many cases are associated with the consumption of milk and undercooked or fermented meats. The stx(2) strains of VTEC O26 seem to be more likely to cause human infections than isolates expressing only stx(1). The isolation and identification of VTEC O26 from foods is labor intensive and time-consuming. We developed a multiplex PCR (M-PCR) assay for the identification and characterization of E. coli O26 VTEC and its detection in raw milk and ground beef. The method is based on the amplification of the wzx, stx(1), and stx(2) genes for the simultaneous detection of the O26 antigen and verocytotoxin types 1 and 2. This M-PCR assay had a sensitivity of 10(8) CFU/ml when applied to a bacterial suspension and of 10(6) CFU/ml or g when applied to both inoculated milk and minced beef samples. This M-PCR assay also was highly specific, and results were consistently negative for negative controls (nonpathogenic E. coli strains, uninoculated milk and beef samples, and samples inoculated with the nontarget microorganisms). This method could be used for the rapid detection of E. coli O26 VTEC from foods and for the rapid identification and characterization of clinical and environmental isolates.

  18. Rapid concentration of bacteria using submicron magnetic anion exchangers for improving PCR-based multiplex pathogen detection.

    PubMed

    Yang, Kun; Jenkins, Daniel M; Su, Wei Wen

    2011-07-01

    Rapid concentration of bacterial targets from dilute solutions to improve subsequent PCR detection is investigated in this study. Submicron (average size 500nm) superparamagnetic anion-exchangers (SiMAG-DEAE) were used successfully to concentrate target bacteria from very dilute solutions. A mass-balance model predicted that for Escherichia coli, the extent of cell concentrating increases almost linearly with increasing sample/SiMAG volume ratio up to about 2000, accompanied by only a slight decrease in the capture efficiency (<10%). Our experimental data generally support this analysis in that the SiMAG beads concentrated bacterial targets by two to three orders of magnitude using a sample/bead volume ratio of about 1000, and lowered the PCR detection limit to a level of 10(2)CFU/mL, from 10(4) to 10(5)CFU/mL without concentrating. Several target bacteria can be concentrated concurrently and detected via multiplex PCR, as illustrated using E. coli and Agrobacterium tumefaciens as model bacteria. Finally, concentration and detection of bacteria in fresh produce samples were demonstrated. The integration of submicron magnetic ion exchangers and PCR detection provides an appealing alternative to immunomagnetic separation/PCR in improving pathogen detection.

  19. Evaluation of Nanogen MGB Alert Detection Reagents in a multiplex real-time PCR for influenza virus types A and B and respiratory syncytial virus.

    PubMed

    Hymas, Weston C; Hillyard, David R

    2009-03-01

    A multiplex real-time RT-PCR assay that detects influenza A, influenza B and respiratory syncytial virus (RSV) using the MGB Alert Influenza A&B/RSV Detection Reagent RUO (Nanogen, San Diego, CA) was developed. The Nanogen detection reagents consist of PCR primers and minor groove binder-conjugated hybridization probes for each virus and an internal control. Virus typing was determined by post-PCR melt curve analysis. A non-competitive armored RNA internal control was co-extracted with each sample to monitor nucleic acid extraction and RT-PCR. The assay was evaluated using a collection of culture, DFA and RT-PCR (Hexaplex, Prodesse, Waukesha, WI) positive and negative samples. The real-time multiplex assay detected 236 of 237 positive specimens for a 99% correlation. Of 30 Hexaplex negative samples tested, the multiplex real-time assay detected an additional 7 positives confirmed using additional PCR assays. Melt curve analysis for each virus produced average melting peaks of 60.4 degrees C, 66.7 degrees C and 69.4 degrees C for influenza A, influenza B and RSV respectively. Sequence analysis of 7 influenza A samples producing aberrant melt curves, confirmed the presence of a single nucleotide polymorphism beneath the influenza A probe. The limit of detection of each virus in 4 different sample types was measured to be between 7 and 806 copies. Overall, the multiplexed real-time RT-PCR assay was sensitive, robust and easy to use.

  20. Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

    PubMed

    Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S

    2015-01-01

    Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

  1. Epidemiology and Clinical Presentations of Respiratory Syncytial Virus Subgroups A and B Detected with Multiplex Real-Time PCR

    PubMed Central

    Liu, Wenkuan; Chen, Dehui; Tan, Weiping; Xu, Duo; Qiu, Shuyan; Zeng, Zhiqi; Li, Xiao; Zhou, Rong

    2016-01-01

    Respiratory syncytial virus (RSV) is one of the most important pathogenic infections of children and requires in-depth research worldwide, and especially in developing countries. We used a novel multiplex real-time PCR to test 5483 patients (≤ 14 years old) hospitalized with respiratory illness in Guangzhou, China, over a 3-year period. Of these patients, 729 were positive for RSV-A (51.2%, 373/729) or RSV-B (48.8%, 356/729), but none was infected with both viruses. Two seasonal peaks in total RSV were detected at the changes from winter to spring and from summer to autumn. RSV-B was dominant in 2013 and RSV-A in 2015, whereas RSV-A and RSV-B cocirculated in 2014. The clinical presentations of 645 RSV-positive patients were analyzed. Bronchiolitis, dyspnea, coryza, vomiting, poor appetite, and diarrhea occurred more frequently in RSV-A-positive than RSV-B-positive patients, whereas chill, headache, myalgia, debility, and rash etc. were more frequent in RSV-B-positive than RSV-A-positive patients, suggesting specific clinical characteristics for RSV-A and RSV-B. Coinfectons with other pathogens were common and diverse. Bronchiolitis, fever (≥ 38°C), and poor appetite were more frequent in patients with single RSV infections than in coinfected patients, suggesting the key pathogenic activity of RSV. Analysis of the relationships between the comparative viral load and clinical presentations showed significant differences in bronchiolitis, fever (≥ 38°C), and rash etc. among patients with different viral loads. This study provides a novel rapid method for detecting RSV subgroups, and provides new insights into the epidemiology and clinical implications of RSV. PMID:27764220

  2. Phylogeographic analysis of hemorrhagic fever with renal syndrome patients using multiplex PCR-based next generation sequencing

    PubMed Central

    Kim, Won-Keun; Kim, Jeong-Ah; Song, Dong Hyun; Lee, Daesang; Kim, Yong Chul; Lee, Sook-Young; Lee, Seung-Ho; No, Jin Sun; Kim, Ji Hye; Kho, Jeong Hoon; Gu, Se Hun; Jeong, Seong Tae; Wiley, Michael; Kim, Heung-Chul; Klein, Terry A.; Palacios, Gustavo; Song, Jin-Won

    2016-01-01

    Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health threat. Next generation sequencing (NGS) is a powerful technology to define genomic sequences of the viruses. Of particular interest is the use of whole genome sequencing (WGS) to perform phylogeographic analysis, that allows the detection and tracking of the emergence of viral infections. Hantaviruses, Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans. We propose to use WGS for the phylogeographic analysis of human hantavirus infections. A novel multiplex PCR-based NGS was developed to gather whole genome sequences of Hantaan virus (HTNV) from HFRS patients and rodent hosts in endemic areas. The obtained genomes were described for the spatial and temporal links between cases and their sources. Phylogenetic analyses demonstrated geographic clustering of HTNV strains from clinical specimens with the HTNV strains circulating in rodents, suggesting the most likely site and time of infection. Recombination analysis demonstrated a genome organization compatible with recombination of the HTNV S segment. The multiplex PCR-based NGS is useful and robust to acquire viral genomic sequences and may provide important ways to define the phylogeographical association and molecular evolution of hantaviruses. PMID:27221218

  3. Phylogeographic analysis of hemorrhagic fever with renal syndrome patients using multiplex PCR-based next generation sequencing.

    PubMed

    Kim, Won-Keun; Kim, Jeong-Ah; Song, Dong Hyun; Lee, Daesang; Kim, Yong Chul; Lee, Sook-Young; Lee, Seung-Ho; No, Jin Sun; Kim, Ji Hye; Kho, Jeong Hoon; Gu, Se Hun; Jeong, Seong Tae; Wiley, Michael; Kim, Heung-Chul; Klein, Terry A; Palacios, Gustavo; Song, Jin-Won

    2016-01-01

    Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health threat. Next generation sequencing (NGS) is a powerful technology to define genomic sequences of the viruses. Of particular interest is the use of whole genome sequencing (WGS) to perform phylogeographic analysis, that allows the detection and tracking of the emergence of viral infections. Hantaviruses, Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans. We propose to use WGS for the phylogeographic analysis of human hantavirus infections. A novel multiplex PCR-based NGS was developed to gather whole genome sequences of Hantaan virus (HTNV) from HFRS patients and rodent hosts in endemic areas. The obtained genomes were described for the spatial and temporal links between cases and their sources. Phylogenetic analyses demonstrated geographic clustering of HTNV strains from clinical specimens with the HTNV strains circulating in rodents, suggesting the most likely site and time of infection. Recombination analysis demonstrated a genome organization compatible with recombination of the HTNV S segment. The multiplex PCR-based NGS is useful and robust to acquire viral genomic sequences and may provide important ways to define the phylogeographical association and molecular evolution of hantaviruses. PMID:27221218

  4. Touchdown nested multiplex PCR detection of Phytophthora cinnamomi and P. cambivora from French and English chestnut grove soils.

    PubMed

    Langrell, Stephen R H; Morel, Olivier; Robin, Cécile

    2011-07-01

    Soil borne Phytophthora cinnamomi and Phytophthora cambivora are considered the most pathogenic species associated with chestnut (Castanea sativa) decline in Europe. Mapping their incidence and distribution from nursery and plantation soils may offer valuable information for limiting spread. As conventional biological baiting and taxonomic confirmation is generally time consuming, labour, logistically and space intensive, we have focused on the development of a specific touchdown nested multiplex Polymerase Chain Reaction (PCR) approach for the simultaneous detection of both species direct from soil. Pre-existing and novel primers, based on Internal Transcribed Spacer (ITS) sequences, have been evaluated for their specificity and use in a multiplex capacity in various combinations. Coupled to this we have modified a mechanical lysis procedure for DNA extraction from up to 10 g of chestnut under storey soils (ranging from 0.5 to 25 μg DNA g(-1)fresh soil). Using serial dilutions and/or polyvinylpolypyrrolidone chromatography purification, both species have been successfully detected, in artificially and naturally infected soils. Levels of assay detection are comparable to other Phytophthora species where PCR based diagnostic systems have been reported. A qualitative evaluation of this approach against conventional baiting is presented.

  5. Multiplex PCR for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels.

    PubMed

    Marangi, Marianna; Giangaspero, Annunziata; Lacasella, Vita; Lonigro, Antonio; Gasser, Robin B

    2015-04-01

    Giardia duodenalis, Cryptosporidium parvum and Toxoplasma gondii are important parasitic protists linked to water- and food-borne diseases. The accurate detection of these pathogens is central to the diagnosis, tracking, monitoring and surveillance of these protists in humans, animals and the environment. In this study, we established a multiplex real-time PCR (qPCR), coupled to high resolution melting (HRM) analysis, for the specific detection and quantification of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). Once optimised, this assay was applied to the testing of samples (n = 232) of treated wastewater and mussels (Mytilus galloprovincialis). Of 119 water samples, 28.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens; of 113 mussel samples, 66.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens, and 13.2% were test-positive for only T. gondii. The specificity of all amplicons produced was verified by direct sequencing. The oo/cysts numbers (per 5 μl of DNA sample) ranged from 10 to 64. The present multiplex assay achieved an efficiency of 100% and a R(2) value of >0.99. Current evidence indicates that this assay provides a promising tool for the simultaneous detection and quantitation of three key protist taxa.

  6. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  7. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  8. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    PubMed Central

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  9. Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay

    PubMed Central

    Queipo-Ortuño, María Isabel; Colmenero, Juan D.; Bermudez, Pilar; Bravo, María José; Morata, Pilar

    2009-01-01

    Background Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp. Methodology Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively. Conclusions The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%–100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis. PMID:19225565

  10. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology.

    PubMed

    Kirkizlar, Eser; Zimmermann, Bernhard; Constantin, Tudor; Swenerton, Ryan; Hoang, Bin; Wayham, Nicholas; Babiarz, Joshua E; Demko, Zachary; Pelham, Robert J; Kareht, Stephanie; Simon, Alexander L; Jinnett, Kristine N; Rabinowitz, Matthew; Sigurjonsson, Styrmir; Hill, Matthew

    2015-10-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer. PMID:26500031

  11. Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus.

    PubMed

    Syrmis, M W; Moser, R J; Whiley, D M; Vaska, V; Coombs, G W; Nissen, M D; Sloots, T P; Nimmo, G R

    2011-12-01

    The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.

  12. Multiplex RT Q-PCR assay for simultaneous quantification of three viruses used for validation of virus clearance by biopharmaceutical production.

    PubMed

    Lute, Scott; Wang, Hua; Sanchez, Davonie; Barletta, Janet; Chen, Qi; Brorson, Kurt

    2009-10-01

    Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log(10) clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6log(10) dynamic range with a limit of detection (LOD) of approximately 1 genome copy/microL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies.

  13. Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

    PubMed

    Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

    2008-01-01

    Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory

  14. Multiplex PMA-qPCR Assay with Internal Amplification Control for Simultaneous Detection of Viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in Environmental Waters.

    PubMed

    Li, Haiyan; Xin, Hongyi; Li, Sam Fong Yau

    2015-12-15

    Pathogenic microorganisms are responsible for many infectious diseases, and pathogen monitoring is important and necessary for water quality control. This study for the first time explored a multiplex quantitative real-time PCR (qPCR) technique combined with propidium monoazide (PMA) to simultaneously detect viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in one reaction from water samples. Sodium lauroyl sarcosinate (sarkosyl) was applied to enhance the dead bacterial permeability of PMA. The sensitivity of the multiplex PMA-qPCR assay achieved two colony-forming units (CFU) per reaction for L. pneumophila and three CFU per reaction for S. typhimurium and S. aureus. No PCR products were amplified from all nontarget control samples. Significantly, with comparable specificity and sensitivity, this newly invented multiplex PMA-qPCR assay took a much shorter time than did conventional culture assays when testing water samples with spiked bacteria and simulated environmental water treatment. The viable multiplex PMA-qPCR assay was further successfully applied to pathogen detection from rivers, canals, and tap water samples after simple water pretreatment. PMID:26512952

  15. Use of multiplexed tandem PCR to estimate the prevalence and intensity of Theileria orientalis infections in cattle.

    PubMed

    Perera, Piyumali K; Gasser, Robin B; Read, Elizabeth; Malmo, Jakob; Nguyen, Hanh; Nyein, Simon; Cheng, Allan; Jex, Aaron R; Rawlin, Grant; Spithill, Terence W; Jabbar, Abdul

    2015-06-01

    This study employed a semi-quantitative, multiplexed tandem PCR (MT-PCR) to assess the prevalence and infection intensity of four genotypes (buffeli, chitose, ikeda and type 5) of Theileria orientalis in cattle in Australia. Genomic DNA samples from blood samples (n=448) collected from 27 to 32 dairy cows from each of 15 dairy herds with a history of recent theileriosis outbreaks (Group 1), and from blood samples available from 24 cows with or without oriental theileriosis (Group 2) were tested using MT-PCR. Results revealed that all four genotypes were present in Group 1 cattle; genotype buffeli had the highest prevalence (80.5%), followed by genotypes ikeda (71.4%), chitose (38.6%) and type 5 (20.3%). Genotype ikeda had the highest average infection intensity in the cattle (relating to 55,277 DNA copies), followed by buffeli, chitose and type 5 (6354-51,648 copies). For Group 2, results indicated that genotype ikeda had a significantly higher average intensity of infection than buffeli in symptomatic cattle (P<0.001), and symptomatic cattle had a higher intensity of ikeda than asymptomatic cattle (P=0.004). Future studies should assess the utility of the present MT-PCR assay as a diagnostic and epidemiological tool in other parts of Australasia and the world.

  16. Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

    PubMed Central

    Ward, Douglas G.; Baxter, Laura; Gordon, Naheema S.; Ott, Sascha; Savage, Richard S.; Beggs, Andrew D.; James, Jonathan D.; Lickiss, Jennifer; Green, Shaun; Wallis, Yvonne; Wei, Wenbin; James, Nicholas D.; Zeegers, Maurice P.; Cheng, KK; Mathews, Glenn M.; Patel, Prashant; Griffiths, Michael; Bryan, Richard T.

    2016-01-01

    Background Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA. Methods DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free). Results Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity). Conclusion This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer. PMID:26901314

  17. Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids

    PubMed Central

    Köller, Thomas; Kurze, Daniel; Lange, Mirjam; Scherdin, Martin; Podbielski, Andreas; Warnke, Philipp

    2016-01-01

    A fully automated multiplex real-time PCR assay—including a sample process control and a plasmid based positive control—for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88–258) copies/ml for HSV1, 171 (CI 95%, 148–194) copies/ml for HSV2 and 84 (CI 95%, 5–163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements. PMID:27092772

  18. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

    PubMed Central

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

  19. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    PubMed

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

  20. Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

    PubMed Central

    Cho, Seongbeom; Boxrud, David J; Bartkus, Joanne M; Whittam, Thomas S; Saeed, Mahdi

    2007-01-01

    Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections. PMID:17692097

  1. Detection of Herpesviridae in whole blood by multiplex PCR DNA-based microarray analysis after hematopoietic stem cell transplantation.

    PubMed

    Debaugnies, France; Busson, Laurent; Ferster, Alina; Lewalle, Philippe; Azzi, Nadira; Aoun, Mickael; Verhaegen, Godelieve; Mahadeb, Bhavna; de Marchin, Jérôme; Vandenberg, Olivier; Hallin, Marie

    2014-07-01

    Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests = 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test.

  2. The prevalence of urogenital micro-organisms detected by a multiplex PCR-reverse line blot assay in women attending three sexual health clinics in Sydney, Australia.

    PubMed

    McKechnie, Michelle L; Hillman, Richard J; Jones, Rachel; Lowe, Penelope C; Couldwell, Deborah L; Davies, Stephen C; King, Fiona; Kong, Fanrong; Gilbert, Gwendolyn L

    2011-07-01

    This study used a previously described multiplex PCR-based reverse line blot (mPCR/RLB) assay to assess the prevalence and distribution of 14 urogenital pathogens or putative pathogens, namely Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Trichomonas vaginalis, Gardnerella vaginalis, Ureaplasma parvum, Ureaplasma urealyticum, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, herpes simplex virus types 1 and 2, and human adenovirus. First-voided urine specimens and endocervical and self-collected vaginal swabs from each of 216 women attending three sexual health clinics in Sydney, Australia, were tested and the results were compared with those of reference methods for each organism. One hundred and sixty-eight women (77.7 %) had at least one and 105 (48.6 %) had more than one target organism, most commonly G. vaginalis and Ureaplasma spp. The prevalence of each of the four known sexually transmissible pathogens was <5 %. Of the 216 women, 111 (51.4 %) reported at least one symptom consistent with genital or urethral infection, including discharge, pain or discomfort. Only G. vaginalis was detected more frequently in women with symptoms (P = 0.05). The specificity of the mPCR/RLB assay compared with that of the reference methods for each organism and for all specimen types was 100 %. The mean sensitivities of the mPCR/RLB assay compared with those of the reference methods for self-collected vaginal swabs, cervical swabs and first-voided urine specimens for all organisms were 99.3, 98.1 and 84.6 %, respectively; however, these differences were not significant. There were no differences in sensitivities between specimen types for C. trachomatis, N. gonorrhoeae, T. vaginalis and H. influenzae, although all were found infrequently. Overall, the mPCR/RLB platform was found to be an accurate testing platform in a sexual health clinic setting.

  3. A PCR amplification method without DNA extraction.

    PubMed

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  4. Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory

    ERIC Educational Resources Information Center

    Elkins, Kelly M.

    2011-01-01

    The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for…

  5. Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

    PubMed

    Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

    2014-04-01

    Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes.

  6. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    PubMed

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.

  7. [Allele polymorphism analysis in coagulation factors F2, F5 and folate metabolism gene MTHFR by using microchip-based multiplex real time PCR].

    PubMed

    Bogdanov, K V; Nikitin, M M; Slyadnev, M N

    2015-01-01

    Single nucleotide polymorphism (SNP) genotyping methods are widely used for the detection of hereditary thrombophilias caused by genetic defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>Т, MTHFR 1298A>C) genes. Moreover, the combination of gene abnormalities in F2 or/and MTHFR with F5 Leiden mutation leads to increased risk of developing thrombosis. Thus, simultaneous detection of the multiple gene mutations in a sample has important clinical relevance. The microchip-based multiplex real time PCR for estimation of allele specific polymorphism in hemostatic and folate metabolism genes presented here has a high efficiency and may be used for laboratory diagnosis. The optimized protocol for estimation of 4 different types of genetic polymorphisms allowed PCR to be performed with minimal quantity of DNA template and PCR reagents including Taq polymerase and a short-term thermocycling. PMID:26215413

  8. Prevalence of Heptavalent Vaccine-related Pneumococcal Serotypes in Nasopharyngeal carrier in children under five years old in Shahrekord, Iran by Multiplex-PCR during 2010- 2011

    PubMed Central

    Rastabi, Reza Imani; Doosti, Abbas; Askari, Shahin; Hafizi, Masoud

    2014-01-01

    Background: Heptavalent pneumococcal vaccine which included pneumococcal serotypes 4, 6B, 9V,14, 18C, 19F and 23F has been regularly used and is effective on preventing invasive pneumococcal infection. This study aimed to determine vaccine-related serotype distribution in nasopharyngeal carrier and healthy children under five years old. Materials and Methods: In this cross-sectional study from September 2010 to September 2011, 363 nasopharyngeal specimens were collected from healthy children in day care centers. In positive cultures of streptococcus pneumoniae (S. pneumonia) distribution, serotypes were detected by Multiplex polymerase chain reaction (PCR). Electrophoresis of PCR products was used for detection of serotypes of S. pneumoniae. Results: The carrier rate of S. pneumoniae was 29.5% with 95% confidence interval as 24.8- 34.5%. Electrophoresis of PCR products for detection of serotypes of S. pneumonia revealed 430, 220, 753, 189, 573, 304, and 384 bp (s) for 4, 6B, 9V, 14, 18C, 19F, and 23F serotypes of S. pneumoniae, respectively. The frequency of 23F, 6B, 19F, and 18C serotypes were 43%, 34%, 18%, and 5% respectively, but other serotypes (4, 9V and 14) were not detected. Conclusion: Based on the 30% carrier rate and high prevalence of most of heptavalent pneumococcal conjugate vaccine serotypes in our study, this vaccine should be used for prevention of invasive infection in Iranian children. PMID:25584276

  9. [Allele polymorphism analysis in coagulation factors F2, F5 and folate metabolism gene MTHFR by using microchip-based multiplex real time PCR].

    PubMed

    Bogdanov, K V; Nikitin, M M; Slyadnev, M N

    2015-01-01

    Single nucleotide polymorphism (SNP) genotyping methods are widely used for the detection of hereditary thrombophilias caused by genetic defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>Т, MTHFR 1298A>C) genes. Moreover, the combination of gene abnormalities in F2 or/and MTHFR with F5 Leiden mutation leads to increased risk of developing thrombosis. Thus, simultaneous detection of the multiple gene mutations in a sample has important clinical relevance. The microchip-based multiplex real time PCR for estimation of allele specific polymorphism in hemostatic and folate metabolism genes presented here has a high efficiency and may be used for laboratory diagnosis. The optimized protocol for estimation of 4 different types of genetic polymorphisms allowed PCR to be performed with minimal quantity of DNA template and PCR reagents including Taq polymerase and a short-term thermocycling.

  10. Design and Development of a Multiplex Real-Time PCR Assay for Detection of HIV-1 and HCV Using Molecular Beacons.

    PubMed

    Paryan, Mahdi; Mohammadi-Yeganeh, Samira; Mirab Samiee, Siamak; Rezvan, Houri

    2012-09-01

    At least 10 million individuals worldwide are co-infected with immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). These two viruses are transmitted most primarily by exposure to infected blood or blood products. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. In the present study, a multiplex real-time PCR assay for simultaneous detection of HCV and HIV-1 using molecular beacons were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'NCR of HCV genome were used for primers and molecular beacon design. The analysis of scalar concentrations of the samples indicated that this multiplex procedure detects at least 1,000 copies/ml of HIV-1 and 100 copies/ml of HCV with linear reference curve (R (2) > 0.94). The results demonstrate that a specificity of 100 % and sensitivity of 96 % can be achieved. The analytical sensitivity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes only detected HIV-1 and all major variants of HCV. This assay may represent an alternative rapid and relatively inexpensive screening method for detection of HIV-1/HCV co-infection especially in blood screening.

  11. Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

    PubMed Central

    Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.

    2013-01-01

    Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002

  12. Assessment of a novel multiplex real-time PCR assay for the detection of the CBPP agent Mycoplasma mycoides subsp. mycoides SC through experimental infection in cattle

    PubMed Central

    2011-01-01

    Background Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP), the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046) and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples. Results The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result. Conclusions The developed multiplex rt-PCR assay is recommended as an efficient tool for rapid confirmation of

  13. Combined stool-based multiplex PCR and microscopy for enhanced pathogen detection in patients with persistent diarrhoea and asymptomatic controls from Côte d'Ivoire.

    PubMed

    Becker, S L; Chatigre, J K; Gohou, J-P; Coulibaly, J T; Leuppi, R; Polman, K; Chappuis, F; Mertens, P; Herrmann, M; N'Goran, E K; Utzinger, J; von Müller, L

    2015-06-01

    Infectious diarrhoea ranks among the leading causes of morbidity worldwide. Although most acute diarrhoeal episodes are self-limiting, the diagnosis and treatment of persistent diarrhoea (≥2 weeks) are cumbersome and require laboratory identification of the causative pathogen. Stool-based PCR assays have greatly improved the previously disappointing pathogen detection rates in high-income countries, but there is a paucity of quality data from tropical settings. We performed a case-control study to elucidate the spectrum of intestinal pathogens in patients with persistent diarrhoea and asymptomatic controls in southern Côte d'Ivoire. Stool samples from 68 patients and 68 controls were obtained and subjected to molecular multiplex testing with the Luminex(®) Gastrointestinal Pathogen Panel (GPP), microscopy and rapid antigen detection tests for the diagnosis of diarrhoeagenic pathogens. Overall, 20 different bacteria, parasites and viruses were detected by the suite of diagnostic methods employed. At least one pathogen was observed in 84% of the participants, and co-infections were observed in >50% of the participants. Enterotoxigenic Escherichia coli (32%), Giardia intestinalis (29%) and Shigella species (20%) were the predominant pathogens, and Strongyloides stercoralis (10%) was the most prevalent helminth. Pathogen frequencies and numbers of co-infections were similar in patients and controls. Although the Luminex(®) GPP detects a broad range of pathogens, microscopy for helminths and intestinal protozoa remains necessary to cover the full aetiological spectrum in tropical settings. We conclude that highly sensitive multiplex PCR assays constitute a useful screening tool, but that positive results might need to be confirmed by independent methods to discriminate active infection from asymptomatic faecal shedding of nucleic acids.

  14. How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection.

    PubMed

    Stockmann, C; Rogatcheva, M; Harrel, B; Vaughn, M; Crisp, R; Poritz, M; Thatcher, S; Korgenski, E K; Barney, T; Daly, J; Pavia, A T

    2015-02-01

    The objective of this study was to compare the aetiologic yield of standard-of-care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal (GI) Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of three (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (p < 0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for Clostridium difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents.

  15. Multiplex PCR in determination of Opiinae parasitoids of fruit flies, Bactrocera sp., infesting star fruit and guava.

    PubMed

    Shariff, S; Ibrahim, N J; Md-Zain, B M; Idris, A B; Suhana, Y; Roff, M N; Yaakop, S

    2014-01-23

    Malaysia is a tropical country that produces commercial fruits, including star fruits, Averrhoa carambola L. (Oxalidales: Oxalidaceae), and guavas, Psidium guajava L. (Myrtales: Myrtaceae). There is a high demand for these fruits, and they are planted for both local consumption and export purposes. Unfortunately, there has been a gradual reduction of these fruits, which has been shown to be related to fruit fly infestation, especially from the Bactrocera species. Most parasitic wasps (Hymenoptera: Braconidae: Opiinae) are known as parasitoids of fruit fly larvae. In this study, star fruits and guavas infested by fruit fry larvae were collected from the Malaysian Agricultural Research and Development Institute. The parasitized larvae were reared under laboratory conditions until the emergence of adult parasitoids. Multiplex PCR was performed to determine the braconid species using two mitochondrial DNA markers, namely cytochrome oxidase subunit I and cytochrome b. Two benefits of using multiplex PCR are the targeted bands can be amplified simultaneously using the same reaction and the identification process of the braconid species can be done accurately and rapidly. The species of fruit flies were confirmed using the COI marker. The results obtained from our study show that Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), Fopius arisanus (Sonan), and Pysttalia incisi (Silvestri) were parasitoids associated with Bactrocera carambolae (Drew and Hancock) (Diptera: Tephritidae) infested star fruits. Fopius arisanus was also the parasitoid associated with Bactrocera papayae (Drew and Hancock) infested guavas. Maximum parsimony was been constructed in Opiinae species to compare tree resolution between these two genes in differentiating among closely related species. The confirmation of the relationship between braconids and fruit fly species is very important, recognized as preliminary data, and highly necessary in biological control programs.

  16. Multiplex PCR in Determination of Opiinae Parasitoids of Fruit Flies, Bactrocera sp., Infesting Star Fruit and Guava

    PubMed Central

    Shariff, S.; Ibrahim, N. J.; Md-Zain, B. M.; Idris, A. B.; Suhana, Y.; Roff, M. N.; Yaakop, S.

    2014-01-01

    Malaysia is a tropical country that produces commercial fruits, including star fruits, Averrhoa carambola L. (Oxalidales: Oxalidaceae), and guavas, Psidium guajava L. (Myrtales: Myrtaceae). There is a high demand for these fruits, and they are planted for both local consumption and export purposes. Unfortunately, there has been a gradual reduction of these fruits, which has been shown to be related to fruit fly infestation, especially from the Bactrocera species. Most parasitic wasps (Hymenoptera: Braconidae: Opiinae) are known as parasitoids of fruit fly larvae. In this study, star fruits and guavas infested by fruit fry larvae were collected from the Malaysian Agricultural Research and Development Institute. The parasitized larvae were reared under laboratory conditions until the emergence of adult parasitoids. Multiplex PCR was performed to determine the braconid species using two mitochondrial DNA markers, namely cytochrome oxidase subunit I and cytochrome b. Two benefits of using multiplex PCR are the targeted bands can be amplified simultaneously using the same reaction and the identification process of the braconid species can be done accurately and rapidly. The species of fruit flies were confirmed using the COI marker. The results obtained from our study show that Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), Fopius arisanus (Sonan), and Pysttalia incisi (Silvestri) were parasitoids associated with Bactrocera carambolae (Drew and Hancock) (Diptera: Tephritidae) infested star fruits. Fopius arisanus was also the parasitoid associated with Bactrocera papayae (Drew and Hancock) infested guavas. Maximum parsimony was been constructed in Opiinae species to compare tree resolution between these two genes in differentiating among closely related species. The confirmation of the relationship between braconids and fruit fly species is very important, recognized as preliminary data, and highly necessary in biological control programs. PMID

  17. Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease.

    PubMed

    Wilkinson, Ray; Wang, Xiangju; Kassianos, Andrew J; Zuryn, Steven; Roper, Kathrein E; Osborne, Andrew; Sampangi, Sandeep; Francis, Leo; Raghunath, Vishwas; Healy, Helen

    2014-01-01

    Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate "real time" gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

  18. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    PubMed Central

    Khairnar, Krishna; Parija, Subhash C

    2007-01-01

    Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens. PMID:17524135

  19. Presence of Undeclared Food Allergens in Cumin: The Need for Multiplex Methods.

    PubMed

    Garber, Eric A E; Parker, Christine H; Handy, Sara M; Cho, Chung Y; Panda, Rakhi; Samadpour, Mansour; Reynaud, Danica H; Ziobro, George C

    2016-02-10

    Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences.

  20. Presence of Undeclared Food Allergens in Cumin: The Need for Multiplex Methods.

    PubMed

    Garber, Eric A E; Parker, Christine H; Handy, Sara M; Cho, Chung Y; Panda, Rakhi; Samadpour, Mansour; Reynaud, Danica H; Ziobro, George C

    2016-02-10

    Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences. PMID:26769163

  1. One-step, multiplex, real-time PCR assay with molecular beacon probes for simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3.

    PubMed

    Besson, Guillaume; Kazanji, Mirdad

    2009-04-01

    A single-tube, multiplex, real-time PCR assay with molecular beacons was established in which various probes were used for the simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3 (HTLV-1, HTLV-2, and HTLV-3, respectively) and of simian T-cell leukemia virus types 1 and 3 (STLV-1 and STLV-3, respectively). The quantitative amplification of the standards with MT4 (HTLV-1) and C19 (HTLV-2) cell lines and a molecular clone of HTLV-3 was linear, with the simplex and multiplex methods having similar efficiencies. A maximum difference of 0.9 (mean, 0.4; range, 0.0 to 0.9) was found between threshold cycle values in single and multiplex reactions. The efficiency with each probe in the multiplex reaction was close to 100%, indicating strong linear amplification. The albumin gene was used to standardize the copy number. Comparable results for the detection and quantification of HTLV-1 were obtained with our new methods and with other real-time PCR methods described previously. With our new multiplex assay, however, we were able to detect and quantify HTLV-2 and -3 and STLV-1 and -3 in clinical specimens, with an excellent dynamic range of 10(6) to 10(0) copies per assay, which the other assays could not do. Thus, it will be possible to determine a wide range of HTLV types in both standard and clinical samples, with a detection of 1 to 10 HTLV copies in samples containing at least 100 cells. Furthermore, our system can provide evidence for multiple infections with the three HTLV types, with separate proviral load results. Our new method also could be used for epidemiological studies in Africa and in countries where HTLVs and STLVs are endemic.

  2. Usefulness of three-channel multiplex real-time PCR and melting curve analysis for simultaneous detection and identification of the Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

    PubMed

    Hong, Yun Ji; Chung, Young Hoon; Kim, Taek Soo; Song, Sang Hoon; Park, Kyoung Un; Yim, Jae Joon; Song, Junghan; Lee, Jae Ho; Kim, Eui Chong

    2011-11-01

    We attempted to determine the benefits of three-channel multiplex real-time PCR and melting curve analysis not only in detecting and distinguishing between nontuberculous mycobacteria (NTM) and the Mycobacterium tuberculosis complex but also in identifying NTM to the species level.

  3. Clinical application of a multiplex real-time PCR assay for simultaneous detection of Legionella species, Legionella pneumophila, and Legionella pneumophila serogroup 1.

    PubMed

    Benitez, Alvaro J; Winchell, Jonas M

    2013-01-01

    We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.

  4. A comparison of flocked swabs and traditional swabs, using multiplex real-time PCR for detection of common gastroenteritis pathogens in Botswana.

    PubMed

    Mokomane, Margaret; Kasvosve, Ishmael; Gaseitsiwe, Simani; Steenhoff, Andrew P; Pernica, Jeffrey M; Lechiile, Kwana; Luinstra, Kathy; Smieja, Marek; Goldfarb, David M

    2016-10-01

    We compared the performance of flocked and matched traditional rectal swabs collected from 236 children admitted with gastroenteritis in Botswana. All samples were tested using real time multiplex-PCR assays for nine enteric pathogens. There was a 20% higher detection of Shigella from flocked swabs, but most other pathogens had similar detection rates. PMID:27460427

  5. Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers.

    PubMed

    Kuwayama, Masaru; Shigemoto, Naoki; Oohara, Sachiko; Tanizawa, Yukie; Yamada, Hiroko; Takeda, Yoshihiro; Matsuo, Takeshi; Fukuda, Shinji

    2011-07-01

    We have developed simultaneous detection of eight genes associated with the five categories of diarrheagenic Escherichia coli by the multiplex PCR assay with Alexa Fluor-labeled primers. This assay can easily distinguish eight genes based on the size and color of amplified products without gel staining.

  6. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    PubMed

    Laude, A; Valot, S; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Le Pape, P; Morio, F

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice.

  7. Thermally multiplexed polymerase chain reaction.

    PubMed

    Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

    2015-07-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  8. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  9. Determination of pig sex in meat and meat products using multiplex real time-PCR.

    PubMed

    Abdulmawjood, A; Krischek, C; Wicke, M; Klein, G

    2012-07-01

    For specific production lines, European retail companies demand exclusively female pork meat. To control the quality of their suppliers the identification and a quantitative detection of the animal sex origin of the meat is therefore of importance for meat processors. To enable a fast and reliable detection of male pig meat, a real time-PCR-system was designed in the present study. This was based on the genes AMEL-X and AMEL-Y. The real time-PCR assay allowed the detection of male pig meat at a concentration of 1% yielding a detection probability of 100% while the detection probability investigating meat samples containing 0.1% male pig meat was 44.4%. The analytic sensitivity of this system was assessed to be <5 pg DNA per PCR reaction. The assessment of the accuracy of the real time-PCR assay to correctly identify sex individuals was investigated with 62 pigs including males (n=29) and females (n=33) belonging to different breeds/lines. With the newly designed test all analysed animals were correctly sexed. No amplification was obtained with cow, goat, sheep, turkey and chicken genomic DNA. The presented assay can be used for sex diagnosis, for the detection of male pig meat and for meat quality control. PMID:22386444

  10. Nested multiplex PCR--a feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root.

    PubMed

    Dong, Xiuli; Zhao, Bin

    2006-08-01

    Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the cornerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5' end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhiza fungal species in a same plant root system. PMID:16989281

  11. Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers.

    PubMed

    Thiede, C; Bornhäuser, M; Oelschlägel, U; Brendel, C; Leo, R; Daxberger, H; Mohr, B; Florek, M; Kroschinsky, F; Geissler, G; Naumann, R; Ritter, M; Prange-Krex, G; Lion, T; Neubauer, A; Ehninger, G

    2001-02-01

    Sequential analysis of chimerism after allogeneic blood stem cell transplantation (BSCT) has been shown to be predictive for graft failure and relapse. We have explored the impact of a novel approach for the quantitative determination of chimerism using a commercial PCR assay with multiplex amplification of nine STR-loci and fluorescence detection. The feasibility was studied in 121 patients transplanted from related or unrelated donors. Follow-up investigation was performed in 88 patients. Twenty-eight of these patients had received a transplantation after dose-reduced conditioning therapy. Results were compared to data obtained by FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors. The analysis was possible in all patients, the median number of informative alleles was 4 (range 1-8) compared to 7 (range 1-9) in the related and unrelated situation, respectively. A good correlation was seen in 84 samples from 14 patients analyzed in parallel with STR-PCR and FISH. Decreasing values of donor chimerism were detected prior to or concomitantly with the occurrence of graft failure and relapse of disease in all patients investigated prospectively. Using FACS-sorted material, eg peripheral blood CD34+ cells, the assay permitted the detection of residual recipient cells with high sensitivity (down to one CD34+ Kasumi cell in 40,000 normal WBC). Evaluation of the inter-laboratory reproducibility revealed that in 20 samples analyzed in three different centers, the median coefficient of variation was 2.1% (range 0.7-9.6%). Taken together, the results support the use of the test as a valuable tool in the follow-up of patients undergoing allogeneic BSCT. In cases lacking PCR-detectable disease-specific gene products, this assay may represent an alternative to recently established real-time PCR methods. PMID:11236950

  12. Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples.

    PubMed

    Swango, Katie L; Hudlow, William R; Timken, Mark D; Buoncristiani, Martin R

    2007-07-20

    Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.

  13. Species specificities among primates probed with commercially available fluorescence-based multiplex PCR typing kits.

    PubMed

    Hiroshige, Yuuji; Ohtaki, Hiroyuki; Yoshimoto, Takashi; Ogawa, Hisae; Ishii, Akira; Yamamoto, Toshimichi

    2015-09-01

    To assess species specificities among primates of signals from short tandem repeat (STR) loci included in two commercially available kits, mainly the AmpFlSTR Identifiler kit and additionally the GenePrint PowerPlex 16 system, we analyzed 69 DNA samples from 22 nonhuman primate species representing apes, Old World Monkeys (OWMs), New World Monkeys (NWMs), and prosimians. Each prosimian species and the NWM cotton-top tamarin apparently lacked all STR loci probed. Only one peak, the amelogenin-X peak, was evident in samples from all other NWMs, except the owl monkey. In contrast, several loci, including the amelogenin-X peak, was evident in samples from each OWM species. Notably, for each ape sample, the amelogenin peaks were concordant with morphological gender of the individual. Among the primates, especially in apes, the numbers of alleles for STR loci were increasing according to their phylogenetic order: prosimiansmultiplex STR kits examined in this study would contribute to forensic examinations. PMID:25899252

  14. Multiplex PCR for the detection of urogenital pathogens in mothers and newborns.

    PubMed

    Nasution, Tetty Aman; Cheong, S F; Lim, C T; Leong, E W K; Ngeow, Y F

    2007-06-01

    Two duplex PCR assays were established for the detection of C. trachomatis (Ct), N. gonorrhoeae (GC), M. hominis (Mh), and U. urealyticum (Uu). These assays were used on clinical specimens obtained from women with Premature Rupture of Membrane or Post Partum Fever, from preterm infants, as well as from women with uneventful pregnancies and their babies delivered vaginally at term. The analytical sensitivity of the duplex PCR assays with internal controls incorporated is 7.0, 19.0, 5x10(3) and 7x10(2) genome copies per reaction for Ct, GC, Mh and Uu respectively. Specificity was demonstrated by the amplification of only target DNA in the presence of other organisms. Among 40 women with normal, at term, deliveries, there were 6 positives for Ct, 2 for GC and 1 for Uu. None of these women had signs of genital tract infection. The Mh/Uu PCR was positive in 11 of 40 PROM cases, with 7 women positive for Uu, 2 for Mh and 2 others for both organisms. Of 40 blood cultures taken from post-partum maternal infections, 6 were positive for Ct and 1 for Mh. Respiratory secretions from 30 premature neonates yielded 5 positives for Uu and one each for Mh and Ct. In contrast, there was only 1 positive result (for Mh) in 30 mature neonates. With 1 exception, all mycoplasma and ureaplasma positives were confirmed by culture and the concordance between paired tracheal aspirates and nasopharyngeal swabs from neonates was 96.7%. These results show the potential use of the duplex PCR assays for the diagnosis of maternal and neonatal disease caused by the four urogenital pathogens.

  15. Y-chromosomal testing of brown bears (Ursus arctos): Validation of a multiplex PCR-approach for nine STRs suitable for fecal and hair samples.

    PubMed

    Aarnes, Siv Grethe; Hagen, Snorre B; Andreassen, Rune; Schregel, Julia; Knappskog, Per M; Hailer, Frank; Stenhouse, Gordon; Janke, Axel; Eiken, Hans Geir

    2015-11-01

    High-resolution Y-chromosomal markers have been applied to humans and other primates to study population genetics, migration, social structures and reproduction. Y-linked markers allow the direct assessment of the genetic structure and gene flow of uniquely male inherited lineages and may also be useful for wildlife conservation and forensics, but have so far been available only for few wild species. Thus, we have developed two multiplex PCR reactions encompassing nine Y-STR markers identified from the brown bear (Ursus arctos) and tested them on hair, fecal and tissue samples. The multiplex PCR approach was optimized and analyzed for species specificity, sensitivity and stutter-peak ratios. The nine Y-STRs also showed specific STR-fragments for male black bears and male polar bears, while none of the nine markers produced any PCR products when using DNA from female bears or males from 12 other mammals. The multiplex PCR approach in two PCR reactions could be amplified with as low as 0.2 ng template input. Precision was high in DNA templates from hairs, fecal scats and tissues, with standard deviations less than 0.14 and median stutter ratios from 0.04 to 0.63. Among the eight di- and one tetra-nucleotide repeat markers, we detected simple repeat structures in seven of the nine markers with 9-25 repeat units. Allelic variation was found for eight of the nine Y-STRs, with 2-9 alleles for each marker and a total of 36 alleles among 453 male brown bears sampled mainly from Northern Europe. We conclude that the multiplex PCR approach with these nine Y-STRs would provide male bear Y-chromosomal specificity and evidence suited for samples from conservation and wildlife forensics.

  16. Y-chromosomal testing of brown bears (Ursus arctos): Validation of a multiplex PCR-approach for nine STRs suitable for fecal and hair samples.

    PubMed

    Aarnes, Siv Grethe; Hagen, Snorre B; Andreassen, Rune; Schregel, Julia; Knappskog, Per M; Hailer, Frank; Stenhouse, Gordon; Janke, Axel; Eiken, Hans Geir

    2015-11-01

    High-resolution Y-chromosomal markers have been applied to humans and other primates to study population genetics, migration, social structures and reproduction. Y-linked markers allow the direct assessment of the genetic structure and gene flow of uniquely male inherited lineages and may also be useful for wildlife conservation and forensics, but have so far been available only for few wild species. Thus, we have developed two multiplex PCR reactions encompassing nine Y-STR markers identified from the brown bear (Ursus arctos) and tested them on hair, fecal and tissue samples. The multiplex PCR approach was optimized and analyzed for species specificity, sensitivity and stutter-peak ratios. The nine Y-STRs also showed specific STR-fragments for male black bears and male polar bears, while none of the nine markers produced any PCR products when using DNA from female bears or males from 12 other mammals. The multiplex PCR approach in two PCR reactions could be amplified with as low as 0.2 ng template input. Precision was high in DNA templates from hairs, fecal scats and tissues, with standard deviations less than 0.14 and median stutter ratios from 0.04 to 0.63. Among the eight di- and one tetra-nucleotide repeat markers, we detected simple repeat structures in seven of the nine markers with 9-25 repeat units. Allelic variation was found for eight of the nine Y-STRs, with 2-9 alleles for each marker and a total of 36 alleles among 453 male brown bears sampled mainly from Northern Europe. We conclude that the multiplex PCR approach with these nine Y-STRs would provide male bear Y-chromosomal specificity and evidence suited for samples from conservation and wildlife forensics. PMID:26264959

  17. Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

    PubMed

    Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

    2014-06-01

    Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen.

  18. Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize.

    PubMed

    Onishi, Mari; Matsuoka, Takeshi; Kodama, Takashi; Kashiwaba, Koichi; Futo, Satoshi; Akiyama, Hiroshi; Maitani, Tamio; Furui, Satoshi; Oguchi, Taichi; Hino, Akihiro

    2005-12-14

    In this study, we developed a novel multiplex polymerase chain reaction (PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within a single reaction. The eight detection primer pairs designed to be construct specific for eight respective GM events (i.e., Bt11, Event176, GA21, MON810, MON863, NK603, T25, and TC1507) and a primer pair for an endogenous reference gene, ssIIb, were included in the nonaplex(9plex) PCR system, and its amplified products could be distinguished by agarose gel and capillary electrophoreses based on their different lengths. The optimal condition enabled us to reliably amplify two fragments corresponding to a construct specific sequence and a taxon specific ssIIb in each of the eight events of GM maize and all of nine fragments in a simulated GM mixture containing as little as 0.25% (w/w) each of eight events of GM maize. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM maize. PMID:16332120

  19. Phylogenetic Analysis of Ara+ and Ara− Burkholderia pseudomallei Isolates and Development of a Multiplex PCR Procedure for Rapid Discrimination between the Two Biotypes

    PubMed Central

    Dharakul, Tararaj; Tassaneetrithep, Boonratn; Trakulsomboon, Suwanna; Songsivilai, Sirirurg

    1999-01-01

    A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B. pseudomallei in an area with endemic melioidosis. This organism is almost identical to B. pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate l-arabinose. These Ara+ isolates are also less virulent than the Ara− isolates in animal models. In addition, clinical isolates of B. pseudomallei available to date are almost exclusively Ara−. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara− and one Ara+) and nine soil isolates (five Ara− and four Ara+) of B. pseudomallei were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara− soil isolates, which were identical to the classical clinical isolates of B. pseudomallei. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara− biotypes. The differences were, however, not sufficient for classification into a new species within the genus Burkholderia. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara− and Ara+ B. pseudomallei isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens. PMID:10325345

  20. Development and Evaluation of a SYBR Green-Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses.

    PubMed

    Chen, Huixin; Parimelalagan, Mariya; Lai, Yee Ling; Lee, Kim Sung; Koay, Evelyn Siew-Chuan; Hapuarachchi, Hapuarachchige C; Ng, Lee Ching; Ho, Phui San; Chu, Justin Jang Hann

    2015-11-01

    Chikungunya virus (CHIKV) and dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. In this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate DENV serotypes 1, 2, 3, and 4 and CHIKV. This SYBR Green I-based one-step multiplex real-time RT-PCR assay is highly sensitive and specific for CHIKV and DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV and to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, and 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- and CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, and 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of DENV and CHIKV in clinical samples.

  1. Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay.

    PubMed

    Zhang, Minxiu; Xie, Zhixun; Xie, Liji; Deng, Xianwen; Xie, Zhiqin; Luo, Sisi; Liu, Jiabo; Pang, Yaoshan; Khan, Mazhar I

    2015-11-01

    A new high-throughput GenomeLab Gene Expression Profiler (GeXP) analyser-based multiplex PCR assay was developed for the detection of eight reproductive and respiratory pathogens in swine. The reproductive and respiratory pathogens include North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), classical swine fever virus (CSFV), porcine circovirus 2 (PCV-2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). Nine pairs of specific chimeric primers were designed and used to initiate PCRs, and one pair of universal primers was used for subsequent PCR cycles. The specificity of the GeXP assay was examined using positive controls for each virus. The sensitivity was evaluated using serial ten-fold dilutions of in vitro-transcribed RNA from all of the RNA viruses and plasmids from DNA viruses. The GeXP assay was further evaluated using 114 clinical specimens and was compared with real-time PCR/single RT-PCR methods. The specificity of the GeXP assay for each pathogen was examined using single cDNA/DNA template. Specific amplification peaks of the reproductive and respiratory pathogens were observed on the GeXP analyser. The minimum copies per reaction detected for each virus by the GeXP assay were as follows: 1000 copies/μl for PRV; 100 copies/μl for CSFV, JEV, PCV-2 and PPV; and 10 copies/μl for SIV-H1, SIV-H3 and PRRSV-NA. Analysis of 114 clinical samples using the GeXP assay demonstrated that the GeXP assay had comparable detection to real-time PCR/single RT-PCR. This study demonstrated that the GeXP assay is a new method with high sensitivity and specificity for the identification of these swine reproductive and respiratory pathogens. The GeXP assay may be adopted for molecular epidemiological surveys of these reproductive and respiratory pathogens in swine populations. PMID:26259690

  2. Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.

    PubMed

    Timmons, Chris; Dobhal, Shefali; Fletcher, Jacqueline; Ma, Li Maria

    2013-04-01

    Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

  3. A new PCR method: one primer amplification of PCR-CTPP products.

    PubMed

    Yin, Guang; Mitsuda, Yoko; Ezaki, Takayuki; Hamajima, Nobuyuki

    2012-10-01

    Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

  4. The polymerase chain reaction (PCR): general methods.

    PubMed

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  5. A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants

    PubMed Central

    Cappy, Pierre; De Oliveira, Fabienne; Gueudin, Marie; Alessandri-Gradt, Elodie

    2016-01-01

    The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns. PMID:26912747

  6. Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis▿†§

    PubMed Central

    Lucignano, Barbara; Ranno, Stefania; Liesenfeld, Oliver; Pizzorno, Beatrice; Putignani, Lorenza; Bernaschi, Paola; Menichella, Donato

    2011-01-01

    Sepsis is a major health problem in newborns and children. Early detection of pathogens allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes. Multiplex PCR has the potential to rapidly identify bloodstream infections, compensating for the loss of blood culture sensitivity. In an Italian pediatric hospital, multiplex PCR (the LightCycler SeptiFast test) was compared to routine blood culture with 1,673 samples obtained from 803 children with suspected sepsis; clinical and laboratory information was used to determine the patient infection status. Excluding results attributable to contaminants, SeptiFast showed a sensitivity of 85.0% (95% confidence interval [CI] = 78.7 to 89.7%) and a specificity of 93.5% (95% CI = 92.1 to 94.7%) compared to blood culture. The rate of positive results was significantly higher with SeptiFast (14.6%) than blood culture (10.3%) (P < 0.0001), and the overall positivity rate was 16.1% when the results of both tests were combined. Staphylococcus aureus (11.6%), coagulase-negative staphylococci (CoNS) (29.6%), Pseudomonas aeruginosa (16.5%), and Klebsiella spp. (10.1%) were the most frequently detected. SeptiFast identified 97 additional isolates that blood culture failed to detect (24.7% P. aeruginosa, 23.7% CoNS, 14.4% Klebsiella spp., 14.4% Candida spp.). Among specimens taken from patients receiving antibiotic therapy, we also observed a significantly higher rate of positivity of SeptiFast than blood culture (14.1% versus 6.5%, respectively; P < 0.0001). On the contrary, contaminants were significantly more frequent among blood cultures than SeptiFast (n = 97 [5.8%] versus n = 26 [1.6%]), respectively; P < 0.0001). SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h. PMID:21471340

  7. Development of a multiplex real-time PCR assay for the detection of Bordetella pertussis and Bordetella parapertussis in a single tube reaction.

    PubMed

    Arbefeville, Sophie; Levi, Michael H; Ferrieri, Patricia

    2014-02-01

    Pertussis is an infectious respiratory disease caused by the fastidious bacterium Bordetella pertussis, which may infect unvaccinated, previously vaccinated children, and adults in whom immunity has waned. Infants are at a particular risk for severe disease and complications. Bordetella parapertussis may cause a similar illness, however the symptoms are less severe and of shorter duration. Pertussis is a highly contagious disease and early diagnosis is essential. Studies have shown that PCR is 2-4 times more likely than culture to detect Bordetella pertussis. We developed a multiplex, real-time PCR assay using analyte-specific reagent (ASR) primers and probes dispensed in a convenient lyophilized bead format that targeted the multi-copy insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively. These specific ASRs were used in conjunction with Cepheid Smartmix. Included in the ASRs is a competitive internal control to evaluate the performance of the PCR reaction. After DNA extraction, amplification and detection were done on the Smart Cycler System, which performs integrated amplification and detection automatically in a single step. Specificity of the assay was confirmed using multiple distinct bacterial strains. Sensitivity of the assay and extraction efficiency were evaluated on DNA isolated from pure bacterial cultures and on spiked respiratory specimens. We also spiked different swab types and transport media to evaluate for interfering substances. To assess accuracy, we studied different patient specimen types received from two outside laboratories that used similar or different methods to detect B. pertussis and B. parapertussis. The sensitivity and the specificity of the assay for B. pertussis were 90% and 96%, respectively, and for B. parapertussis 71% (only 7 positive specimens were available for testing) and 100%, respectively. Our assay was found to be a valid method for the simultaneous detection of B. pertussis and B

  8. Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis.

    PubMed

    Chen, Huang; Zhu, Zaifang; Lu, Joann Juan; Liu, Shaorong

    2015-02-01

    Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary-hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions. PMID:25555111

  9. The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform

    PubMed Central

    Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

    2016-01-01

    Introduction Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analys