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Sample records for quantitative real-time reverse

  1. Analysis of liver connexin expression using reverse transcription quantitative real-time polymerase chain reaction

    PubMed Central

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283

  2. Evaluation of various real-time reverse transcription quantitative PCR assays for norovirus detection.

    PubMed

    Yoo, Ju Eun; Lee, Cheonghoon; Park, SungJun; Ko, GwangPyo

    2017-02-01

    Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for sensitive and accurate detection for these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assay A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, as well as sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A Zen internal quencher, which decreases nonspecific fluorescence during the PCR reaction, was added to Assay D's probe which further improved assay performance. This study compared several detection assays for noroviruses and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

  3. The transcription analysis of duck enteritis virus UL49.5 gene using real-time quantitative reverse transcription PCR.

    PubMed

    Lin, Meng; Jia, Renyong; Wang, Mingshu; Gao, Xinghong; Zhu, Dekang; Chen, Shun; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue; Cheng, Anchun

    2013-10-01

    Duck enteritis virus (DEV) UL49.5 encoding glycoprotein N was a conserved gene. The transcription dynamic process of UL49.5 homologous genes in herpesviruses was reported. However, the transcription dynamic process of DEV UL49.5 gene has not yet been established. In this study, a real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay was established to test the transcription dynamic process of DEV UL49.5 gene, and the recombinant plasmid pUCm-T/UL49.5 was constructed as the standard DNA. The samples prepared from DEV-infected (at different time points) and uninfected cell were detected and calculated. The results demonstrated that the real-time qRT-PCR assay was successfully established. The transcription product of DEV UL49.5 gene was first detected at 0.5 h post infection (p.i.), increased at 8 h p.i. and reached a peak at 60 h p.i. Our results illustrated that DEV UL49.5 gene could be regarded as a late gene. The transcription dynamic process of DEV UL49.5 gene may provide a significant clue for further studies of DEV UL49.5 gene.

  4. Quantitative real-time imaging of glutathione

    USDA-ARS?s Scientific Manuscript database

    Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

  5. Quantitative Real-Time Reverse Transcription-PCR Analysis of Deformed Wing Virus Infection in the Honeybee (Apis mellifera L.)

    PubMed Central

    Chen, Y. P.; Higgins, J. A.; Feldlaufer, M. F.

    2005-01-01

    Deformed wing virus (DWV) can cause wing deformity and premature death in adult honeybees, although like many other bee viruses, DWV generally persists as a latent infection with no apparent symptoms. Using reverse transcription (RT)-PCR and Southern hybridization, we detected DWV in all life stages of honeybees, including adults with and without deformed wings. We also found DWV in the parasitic mite Varroa destructor, suggesting that this mite may be involved in the transmission of DWV. However, the detection of the virus in life stages not normally associated with mite parasitism (i.e., eggs and larvae) suggests that there are other modes of transmission. The levels of DWV in different life stages of bees were investigated by using TaqMan real-time quantitative RT-PCR. The amounts of virus varied significantly in these different stages, and the highest levels occurred in pupae and in adult worker bees with deformed wings. The variability in virus titer may reflect the different abilities of bees to resist DWV infection and replication. The epidemiology of DWV is discussed, and factors such as mite infestation, malnutrition, and climate are also considered. PMID:15640219

  6. Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

    PubMed Central

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

    2006-01-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

  7. Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine.

    PubMed

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Angel; Mas, Albert; Guillamón, Jose M

    2006-11-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

  8. Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

    USDA-ARS?s Scientific Manuscript database

    A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

  9. A novel duplex real time quantitative reverse transcription polymerase chain reaction for rubella virus with armored RNA as a noncompetitive internal positive control.

    PubMed

    Zhao, Lihong; Li, Ruiying; Liu, Aihua; Zhao, Shuping

    2015-07-01

    The objective of this study was to build and apply a duplex real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus. Firstly, a 60-bp-long armored RV RNA was constructed in the laboratory. Secondly, a duplex real time RT-PCR assay was established. Thirdly, the 60-bp-long armored RV RNA was used as an internal positive control (IPC) for the duplex real time RT-PCR. And finally the duplex real time RT-PCR assay was applied to detect RV RNA in clinical specimens. The in-house assay has a high amplification efficiency (0.99), a high analytical sensitivity (200 copies/mL), and a good reproducibility. The diagnostic specificity and sensitivity of the in-house assay were both 100%, due to the monitoring of the armored RV RNA IPC. Therefore, the in-house duplex real time quantitative RT-PCR assay is a specific, sensitive, reproducible and accurate assay for quantitation of RV RNA in clinical specimens. And noncompetitive armored RV RNA IPC can monitor RT-PCR inhibition and prevent false-negative and inaccurate results in the real time detection system.

  10. Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus.

    PubMed

    Zhu, Xiangdong; Wang, Yufei; Chen, Jianfei; Zhang, Xin; Shi, Hongyan; Shi, Da; Gao, Jing; Feng, Li

    2016-08-01

    Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV.

  11. [Determination of the biological attribute of evidence at the scene using reverse transcription PCR and real-time fluorescent quantitative PCR].

    PubMed

    Xu, Yan; Zhang, Chen; Xu, Qing-Wen; Huang, Jiang-Ping; Liu, Ya-Nan; Zou, Kai-Nan; Ping, Yuan; Zhou, Huai-Gu

    2013-08-01

    To explore the feasibility of biological method to identify the biological attribute of samples at crime scene. Thirty samples of ten blood stains, ten saliva stains and ten semen stains were selected, and all the samples were processed by the routine method and biomolecular method, respectively. Both RNA and DNA were isolated using DNA-RNA co-extraction technology and the mRNA was converted into cDNA using reverse transcription PCR (RT-PCR). Three pairs of specific primers were designed for blood stain, saliva stain and semen stain based on the different target genes in three specific tissues and the primers were amplified using real-time fluorescent quantitative PCR. The differences in these biological samples were evaluated by melting temperature (Tm) values and the size of the amplification fragment. The Tm values of blood stain, saliva stain and semen stain were (84.5 +/- 0.2) degrees C, (76.9 +/- 0.3) degrees C and (88.5 +/- 0.2) degrees C, respectively. The length of PCR fragments of them was 177bp, 134bp and 294bp, respectively. Compared with the routine method, RT-PCR with real-time fluorescent quantitative PCR is highly specific, sensitive and reliable to identify the biological attribute of evidence, and can be potentially applied to determine evidence attribute in forensic practice.

  12. Quantitative Real-Time PCR: Recent Advances.

    PubMed

    Singh, Charanjeet; Roy-Chowdhuri, Sinchita

    2016-01-01

    Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in real time in a "closed-tube" system. Although this technique can provide an absolute quantification of the initial template copy number, quantification relative to a control sample or second sequence is typically adequate. The quantification process employs melting curve analysis and/or fluorescent detection systems and can provide amplification and genotyping in a relatively short time. Here we describe the properties and uses of various fluorescent detection systems used for quantification.

  13. Use of real-time quantitative reverse transcription polymerase chain reaction for the detection of African horse sickness virus replication in Culicoides imicola.

    PubMed

    Scheffer, Elisabeth G; Venter, Gert J; Joone, Christopher; Osterrieder, Nikolaus; Guthrie, Alan J

    2011-11-11

    Despite its important role as vector for African horse sickness virus (AHSV), very little information is available on the dissemination of this virus in Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae). This study reports on the applicability of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect AHSV in dissected midges. A total of 96 midges were fed on AHSV-infected blood, after which one test group was dissected into head/thorax and abdomen segments immediately after feeding and the other only after 10 days of incubation. The majority of the midges (96%) ingested the virus successfully and there was no significant difference between the virus concentration in the heads/thoraxes and the abdomens immediately after feeding. After incubation, virus was detected in 51% of the midges and it was confined to the abdomen in the majority of these. The fact that virus was detected only in the heads/thoraxes of four Culicoides midges after incubation suggests the presence of a mesenteronal escape barrier. Replication in the salivary glands was not shown. An increase of the mean virus concentration in the abdomen after incubation indicates localised viral replication. The real-time RT-qPCR is recommended for further studies investigating the replication and dissemination of AHSV in Culicoides midges.

  14. SYBR(®) Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses.

    PubMed

    Poojari, Sudarsana; Alabi, Olufemi J; Okubara, Patricia A; Naidu, Rayapati A

    2016-09-01

    A SYBR(®) Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt-curve analysis (MCA) was optimized for the detection of nine grapevine viruses. The detection limits for simplex qRT-PCR for all nine grapevine viruses were estimated to be in the range of 214-1112 copies of the virus genome. Amplicons with melting temperatures (Tm) separated by at least 2°C in the MCA could differentiate two viruses in the same reaction. Therefore, eight of the nine viruses could be co-diagnosed in five different combinations of duplex assays. Of 305 grape leaf samples from the field or greenhouse, 162 were positive for at least one of the nine grapevine viruses using the duplex qRT-PCR assays. In contrast, only 127 samples were positive using endpoint RT-PCR and PCR assays, indicating the enhanced sensitivity of duplex real-time PCR. In addition, the duplex qRT-PCR assays were be used to detect Grapevine leafroll associated virus 3 (GLRaV-3) in its vector, the grape mealybug (Pseudococcus maritimus Ehrhorn), and Grapevine red blotch-associated virus (GRBaV) in Virginia creeper leafhopper (Erythroneura ziczac Walsh). The simplex and duplex real-time PCR assays developed in this study can be used to examine transmission of co-occruing viruses by insect vectors as well as for rapid and sensitive detection of viruses in infected grapevines. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus.

    PubMed

    Wei, Haiyan; Zeng, Jing; Deng, Congliang; Zheng, Chengzhong; Zhang, Ximeng; Ma, Dan; Yi, Yong

    2013-03-01

    A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ∼100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.

  16. Assessment of environmental factors on Listeria monocytogenes Scott A inlA gene expression by relative quantitative Taqman real-time reverse transcriptase PCR.

    PubMed

    Hanna, Scott E; Wang, Hua H

    2006-11-01

    Several virulence factors are involved in Listeria monocytogenes pathogenicity. L. monocytogenes internalins, particularly internalin A, are required for bacterial adhesion to and invasion of human intestinal epithelial cells. The expression of internalins is thus related to virulence. Identification of conditions involved in regulating the expression of L. monocytogenes virulence factors is essential for developing targeted strategies to control listeriosis incidence and improving therapeutic approaches. The primary aim of this study was to develop a quantitative real-time reverse transcriptase PCR platform to study the impact of environmental factors on L. monocytogenes Scott A virulence factor expression, particularly in potentially complex ecosystems. A Taqman PCR-based, rapid quantitative gene expression evaluation method was established with the L. monocytogenes ribosomal protein L4 encoding gene used as an internal standard. Our data suggest that inlA expression is influenced by food composition and temperature, indicating that certain food processing or storage conditions, such as the use of lactic and acetic acids at common storage temperatures, could affect the expression of L. monocytogenes virulence factor.

  17. Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT.

    PubMed

    Luchsinger, Charlotte; Arias, María Elena; Vargas, Tamara; Paredes, Marcos; Sánchez, Raúl; Felmer, Ricardo

    2014-11-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive and accurate tool for quantitative estimation of gene transcription levels in preimplantation embryos. To control for possible experimental variations, gene expression data must be normalized using internal control genes commonly known as reference genes. However, the stability of reference genes can vary depending on the state of development and/or experimental conditions; hence the assessment of their stability is essential before initiating a gene expression analysis. In the present study, we used RT-qPCR to measure the transcript levels of 10 commonly used reference genes and analyzed their expression stability in bovine blastocysts produced by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). Using the geNorm program, we found the best combination of genes to normalize gene expression data in bovine embryos at the blastocyst stage produced by IVF (HMBS, SF3A1, and HPRT1), ICSI (H2A, HMBS, and GAPDH), SCNT (ACTB, SF3A1, and SDHA) and/or between blastocysts produced by these methods (GAPDH, HMBS and EEF1A2). We also demonstrated that not only the culture conditions may affect the expression patterns in bovine blastocysts but also the choice of embryo production method may have an important effect.

  18. Quantitation of cytosine deaminase mRNA by real-time reverse transcription polymerase chain reaction: a sensitive method for assessing 5-fluorocytosine toxicity in vitro.

    PubMed

    Miller, C Ryan; Gustin, Allen N; Buchsbaum, Donald J; Vickers, Selwyn M; Manne, Upender; Grizzle, William E; Cloud, Gretchen A; Diasio, Robert B; Johnson, Martin R

    2002-02-15

    Cytosine deaminase/5-fluorocytosine (CD/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), semiquantitative immunocytochemistry (ICC), and 5-[(3)H]FC enzyme assay, respectively. Results with human colon (LS174T) cancer cells infected with a replication-incompetent adenovirus encoding CD (AdCMVCD) demonstrated a significant correlation between CD mRNA and enzyme activity up to 24 h postinfection. A direct correlation was found between CD dose (AdCMVCD PFU/cell) and CD mRNA and protein expression (P < 0.002) in both LS174T and BxPC-3 pancreatic cancer cells, but the relationship with enzyme activity was less strong in LS174T cells (P = 0.09). A remarkable concordance existed among Q-RT-PCR, ICC and enzyme assays with both cell lines. Importantly, CD dose and mRNA and protein expression inversely correlated with 5-FC IC(50) (P < 0.02). Quantitation of CD markers also facilitated identification of factors governing differential susceptibility to CD/5-FC. These results suggest that Q-RT-PCR will be useful for monitoring transgene expression in future studies using improved CD-based expression vectors and may also be useful in predicting the response to CD/5-FC therapy, which is likely to be heterogeneous in the patient population.

  19. Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

    USDA-ARS?s Scientific Manuscript database

    Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

  20. Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

    USDA-ARS?s Scientific Manuscript database

    Efforts to analyze the replicative RNA produced by Maize fine streak virus (MFSV) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

  1. Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenic Bacillus thuringiensis isolates.

    PubMed

    Kaminska, Paulina S; Yernazarova, Aliya; Murawska, Emilia; Swiecicki, Jakub; Fiedoruk, Krzysztof; Bideshi, Dennis K; Swiecicka, Izabela

    2014-08-01

    Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

  2. Identification and Evaluation of Suitable Reference Genes for Gene Expression Studies in the Whitefly Bemisia tabaci (Asia I) by Reverse Transcription Quantitative Real-Time PCR

    PubMed Central

    Collins, Carl; Patel, Mitulkumar V.; Colvin, John; Bailey, David; Seal, Susan

    2014-01-01

    This study presents a reliable method for performing reverse transcription quantitative real-time PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data presented here will assist future transcript profiling studies in whiteflies. PMID:25373210

  3. Real-Time Quantitative Reverse Transcription PCR for Monitoring of Blood-Stage Plasmodium falciparum Infections in Malaria Human Challenge Trials

    PubMed Central

    Murphy, Sean C.; Prentice, Jennifer L.; Williamson, Kathryn; Wallis, Carolyn K.; Fang, Ferric C.; Fried, Michal; Pinzon, Cris; Wang, Ruobing; Talley, Angela K.; Kappe, Stefan H. I.; Duffy, Patrick E.; Cookson, Brad T.

    2012-01-01

    To detect pre-patent parasitemia, we developed a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the asexual 18S ribosomal RNA (rRNAs) of Plasmodium falciparum. Total nucleic acids extracted from whole blood were combined with control RNA and tested by qRT-PCR. The assay quantified > 98.7% of parasite-containing samples to ±0.5 log10 parasites/mL of the nominal value without false positives. The analytical sensitivity was ≥ 20 parasites/mL. The coefficient of variation was 0.6% and 1.8% within runs and 1.6% and 4.0% between runs for high and low parasitemia specimens, respectively. Using this assay, we determined that A-type 18S rRNAs are stably expressed at 1×104 copies per ring-stage parasite. When used to monitor experimental P. falciparum infection of human volunteers, the assay detected blood-stage infections 3.7 days earlier on average than thick blood smears. This validated, internally controlled qRT-PCR method also uses a small (50 μL) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials. PMID:22403305

  4. Near Real Time Quantitative Gas Analysis Techniques

    NASA Astrophysics Data System (ADS)

    Herget, William F.; Tromp, Marianne L.; Anderson, Charles R.

    1985-12-01

    A Fourier transform infrared (FT-IR) - based system has been developed and is undergoing evaluation for near real time multicomponent quantitative analysis of undiluted gaseous automotive exhaust emissions. The total system includes: (1) a gas conditioning system (GCS) for tracer gas injection, gas mixing, and temperature stabilization; and (2) an exhaust gas analyzer (EGA) consisting of a sample cell, an FT-IR system, and a computerized data processing system. Tests have shown that the system can monitor about 20 individual species (concentrations down to the 1-20 ppm range) with a time resolution of one second. Tests have been conducted on a chassis dynamometer system utilizing different autos, different fuels, and different driving cycles. Results were compared with those obtained using a standard constant volume sampling (CVS) system.

  5. Optimization of Reference Genes for Normalization of Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction Results in Senescence Study of Mesenchymal Stem Cells.

    PubMed

    Su, Xiaodong; Yao, Xinglei; Sun, Zhao; Han, Qin; Zhao, Robert Chunhua

    2016-09-15

    Recently, it has been suggested that cellular senescence is associated with stem cell exhaustion, which reduces the regenerative potential of tissues and contributes to aging and age-related diseases. Mesenchymal stem cells (MSCs) attract a large amount of attention in stem cell research and regeneration medicine because they possess multiple advantages and senescent MSCs could be one of the most useful stem cell models in aging studies. It is important to quantitatively evaluate senescence markers to both identify and study the mechanisms involved in MSC senescence. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the most widely used tool to quantify the mRNA levels of markers. However, no report has demonstrated the optimal reference genes that should be used to normalize RT-qPCR in senescence studies of MSCs. In this study, we compared 16 commonly used reference genes (GAPDH, ACTB, RPL13A, TBP, B2M, GUSB, RPLPO, YWHAZ, RPS18, EEF1A1, ATP5F1, HPRT1, PGK1, TFRC, UBC, and PPIA) in proliferating or replicative-senescent human adipose-derived MSCs (hAD-MSCs) that were isolated from seven healthy donors aged 29-59 years old. Three algorithms (geNorm, NormFinder, and BestKeeper) were used to determine the most optimal reference gene. The results showed that PPIA exhibited the most stable expression during senescence, while the widely used ACTB exhibited the lowest stability. We also confirmed that different reference genes lead to different evaluations of senescence markers. Our work ensures that results obtained from senescence studies of hAD-MSCs will be appropriately evaluated in both basic research and clinical trials.

  6. Detection of the pandemic H1N1/2009 influenza A virus by a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction assay.

    PubMed

    Yang, Zhu; Mao, Guoliang; Liu, Yujun; Chen, Yuan-Chuan; Liu, Chengjing; Luo, Jun; Li, Xihan; Zen, Ke; Pang, Yanjun; Wu, Jianguo; Liu, Fenyong

    2013-02-01

    A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

  7. Selection and validation of reference genes for normalization of quantitative real-time reverse transcription PCR analysis in Poria cocos (Schw.) Wolf (Fuling).

    PubMed

    Zhang, Xin; Xu, Zhi-Chao; Xu, Jiang; Ji, Ai-Jia; Luo, Hong-Mei; Song, Jing-Yuan; Sun, Chao; Hu, Yuan-Lei; Chen, Shi-Lin

    2016-01-01

    Quantitative real-time reverse transcription PCR (qRT-PCR) requires a stable internal control to avoid misinterpretation of data or errors for gene expression normalization. However, there are still no validated reference genes for stable internal control in Poria cocos (Schw.) Wolf (Fuling). This study aims to validate the reference genes of P. cocos. This study firstly collected the 14 candidate reference genes by BLASTP from the genome of P. cocos for qRT-PCR analysis to determine the expression levels of 14 housekeeping genes (GAPDH, MAPK, β-Act, RPB2, RPB1-1, RPB1-2, his3-1, his3-2, APT, SAMDC, RP, β-Tub, EIF, and CYP) under different temperatures and in response to different plant hormones (indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid), and the threshold cycle (Ct) values. The results were analyzed by four programs (i.e., geNorm, NormFinder, BestKeeper, and RefFinder) for evaluating the candidate reference genes. SAMDC, his3-2, RP, RPB2, and his3-1 were recommended as reference genes for treating P. cocos with indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid, respectively. Under different temperatures RPB2 was the most stable reference gene. CYP was the most stable gene for all 90 samples by RefFinder. SAMDC, his3-2, RP, RPB2, and his3-1 were evaluated to be suitable reference genes for P. cocos following different treatments. RPB2 was the most stable reference gene under different temperatures and CYP was the most stable gene in the mycelia under all six evaluated conditions.

  8. Real-time quantitative reverse transcription-PCR analysis of expression stability of Aggregatibacter actinomycetemcomitans fimbria-associated gene in response to photodynamic therapy.

    PubMed

    Pourhajibagher, Maryam; Monzavi, Abbas; Chiniforush, Nasim; Monzavi, Mohammad Moein; Sobhani, Shaghayegh; Shahabi, Sima; Bahador, Abbas

    2017-06-01

    Aggregatibacter actinomycetemcomitans is an etiological agent of both chronic and aggressive periodontitis. Dissemination of A. actinomycetemcomitans from the oral cavity and initiation of systemic infections has led to new approaches for treatment being needed. In this study, a series of experiments presented investigated the effect of methylene blue (MB)-mediated antimicrobial photodynamic therapy (aPDT) on cell viability and expression of fimbria-associated gene (rcpA) in A. actinomycetemcomitans. To determine the dose-depended effects of aPDT, A. actinomycetemcomitans ATCC 33384 strain photosensitized with MB was irradiated with diode laser following bacterial viability measurements. Cell-surviving assay and expression ratio of rcpA were assessed by colony forming unit and real-time quantitative reverse transcription-PCR (qRT-PCR) assays, respectively. In the current study, MB-mediated aPDT using 100μg/mL showed significant reduction in A. actinomycetemcomitans growth when compared to the control (P<0.05). Sub-lethal dose of aPDT against A. actinomycetemcomitans was 25μg/mL MB at fluency of 93.75J/cm(2). Sub-lethal dose of aPDT could lead to about four-fold suppression of expression of rcpA. High doses of MB-mediated aPDT could potentially exhibit antimicrobial activity, and the expression of rcpA as an important virulence factor of this strain is reduced in cells surviving aPDT with MB. So, aPDT can be a valuable tool for the treatment of A. actinomycetemcomitans infections. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature.

    PubMed

    Chen, Ying; Korkeala, Hannu; Lindén, Jere; Lindström, Miia

    2008-10-01

    The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30 degrees C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10 degrees C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30 degrees C. The maximum botE expression levels and average neurotoxin levels at 10 degrees C were 45 to 65% of those at 30 degrees C. The relative mRNA levels at 10 degrees C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30 degrees C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30 degrees C, whereas at 10 degrees C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

  10. Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search

    PubMed Central

    2013-01-01

    Background The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated. We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system – specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. Results We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the

  11. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan).

    PubMed

    Saponari, Maria; Manjunath, Keremane; Yokomi, Raymond K

    2008-01-01

    A quantitative and multiplex real-time RT-PCR assay was developed to detect Citrus tristeza virus (CTV) along with plant mRNA, which serves as an internal control to ascertain RNA extraction quality. The real-time technique was validated against 39 CTV strains from around the world as well as with the aphid vector, Aphis gossypii, given a 48 h acquisition access period on a CTV source plant. The assay was effective for quantitation of the viral template in infected plants and in single aphids. CTV detection was compared from different plant tissues and for different RNA isolation methods from aphids. Less than 1 fg was consistently detected when RNA transcripts were diluted in extracts from healthy plants while RNA copies carried by single aphids were estimated to be between 12,000 and 13,000,000. The assay was more sensitive and less time consuming than ELISA or traditional RT-PCR. The real-time RT-PCR assay developed is a valuable new tool for detection and titer quantitation of CTV.

  12. Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen Cryptosporidium parvum In Vitro

    PubMed Central

    Cai, Xiaomin; Woods, Keith M.; Upton, Steve J.; Zhu, Guan

    2005-01-01

    We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (CT[P18S]) and host cells (CT[H18S]), and evaluates the relative expression between parasite and host rRNA levels (i.e., ΔCT = CT[P18S] − CT[H18S]) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (ΔΔCT) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in ∼3 h) in dead parasites is faster than that of DNA (99% in ∼24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 μg/ml and 89.7 to 119.0 μg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets. PMID:16251280

  13. Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.

    PubMed

    Modesto, P; Peletto, S; Pisoni, G; Cremonesi, P; Castiglioni, B; Colussi, S; Caramelli, M; Bronzo, V; Moroni, P; Acutis, P L

    2013-01-01

    Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal

  14. New in situ capture quantitative (real-time) reverse transcription-PCR method as an alternative approach for determining inactivation of Tulane virus.

    PubMed

    Wang, Dapeng; Xu, Shuxia; Yang, David; Young, Glenn M; Tian, Peng

    2014-04-01

    Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrated that this in situ capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively

  15. Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens.

    PubMed

    Southern, Timothy R; Racsa, Lori D; Albariño, César G; Fey, Paul D; Hinrichs, Steven H; Murphy, Caitlin N; Herrera, Vicki L; Sambol, Anthony R; Hill, Charles E; Ryan, Emily L; Kraft, Colleen S; Campbell, Shelley; Sealy, Tara K; Schuh, Amy; Ritchie, James C; Lyon, G Marshall; Mehta, Aneesh K; Varkey, Jay B; Ribner, Bruce S; Brantly, Kent P; Ströher, Ute; Iwen, Peter C; Burd, Eileen M

    2015-09-01

    Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 10(7) to 4 × 10(2) 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 10(2) TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.).

    PubMed

    Galeano, Esteban; Vasconcelos, Tarcísio Sales; Ramiro, Daniel Alves; De Martin, Valentina de Fátima; Carrer, Helaine

    2014-07-22

    Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. This study proposes a first broad

  17. Detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-PCR and correlation with virus detection in embryonated eggs.

    PubMed

    Roh, Ha-Jung; Hilt, Deborah A; Jackwood, Mark W

    2014-09-01

    Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We

  18. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

    PubMed Central

    2014-01-01

    Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study

  19. Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription-quantitative real-time PCR.

    PubMed

    Ananthakrishnan, G; Venkataprasanna, T; Roy, Avijit; Brlansky, R H

    2010-03-01

    A multiplex real-time PCR assay was developed to detect and quantify the Citrus tristeza virus (CTV) genotypic mixture present in infected plants. CTV isolate FS627, a complex Florida isolate containing T36, T30 and VT genotypes and its aphid transmitted subisolates was used. The relative quantitative assay was carried out using specific primers and probes developed from the genotypes of three CTV virus isolates and included the coat protein region of isolate T36 and the 5' end, ORF 1a and ORF 2 region of isolates T36, T30 and VT. Among the three genotypes present in the aphid transmitted subisolates, the T30 genotype showed higher overall relative quantitation in all specific regions compared to other isolates. The profiles of the some aphid transmitted subisolates were different from the parent source from which they transmitted. The 2(-DeltaDeltaCt) method (the amount of target, normalized to an endogenous control and relative to a calibrator) was used to analyze the relative titers of the three reference genotypes in the aphid transmitted plants infected with FS627. This protocol enabled assessments of CTV genetic diversity in the aphid transmitted subisolates. This simple quantitative assay was sensitive, efficient, and took less time than other existing methods. This relative quantitative assay will be a reliable tool for diagnosis, detection and genetic diversity studies on CTV. Published by Elsevier B.V.

  20. Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results

    PubMed Central

    Garcia-Bardon, Andreas

    2016-01-01

    Real-time reverse transcription polymerase chain reaction (PCR) is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups. PMID:27898720

  1. Detection and quantitation of infectious pancreatic necrosis virus by real-time reverse transcriptase-polymerase chain reaction using lethal and non-lethal tissue sampling.

    PubMed

    Bowers, Robert M; Lapatra, Scott E; Dhar, Arun K

    2008-02-01

    Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabirnavirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR((R)) Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout beta-actin and elongation factor 1alpha (EF-1alpha) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84 degrees C for IPNV NS, trout beta-actin, and EF-1alpha genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 10(7) RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin

  2. A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.

    PubMed

    Maan, Narender S; Maan, Sushila; Belaganahalli, Manjunatha; Pullinger, Gillian; Montes, Antonio J Arenas; Gasparini, Marcela R; Guimera, Marc; Nomikou, Kyriaki; Mertens, Peter P C

    2015-03-01

    Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes). Copyright © 2014. Published by Elsevier B.V.

  3. Detection of live Salmonella sp. cells in produce by a TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA.

    PubMed

    González-Escalona, Narjol; Hammack, Thomas S; Russell, Mindi; Jacobson, Andrew P; De Jesús, Antonio J; Brown, Eric W; Lampel, Keith A

    2009-06-01

    Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 10(3) CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 10(5) and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/ approximately ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers.

  4. Validation and Application of a Commercial Quantitative Real-Time Reverse Transcriptase-PCR Assay in Investigation of a Large Dengue Virus Outbreak in Southern Taiwan

    PubMed Central

    Tsai, Huey-Pin; Tsai, You-Yuan; Lin, I-Ting; Kuo, Pin-Hwa; Chang, Kung-Chao; Chen, Jung-Chin; Ko, Wen-Chien; Wang, Jen-Ren

    2016-01-01

    Background Accurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015. Methods Sera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an “in-house” qualitative DENV-specific RT-PCR assay. Results A total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 102 and 106 copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 106–109 copies/mL, which was markedly higher than the rate observed in the other age groups. Conclusions The LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates. PMID:27732593

  5. [Research progress of real-time quantitative PCR method for group A rotavirus detection].

    PubMed

    Guo, Yan-Qing; Li, Dan-Di; Duan, Zhao-Jun

    2013-11-01

    Group A rotavirus is one of the most significant etiological agents which causes acute gastroenteritis among infants and young children worldwide. So far, several method which includes electron microscopy (EM), enzyme immunoassay (EIA), reverse transcription-polymerase chain reaction (RT-PCR)and Real-time Quantitative PCR has been established for the detection of rotavirus. Compared with other methods, Real-time quantitative PCR have advantages in specificity, sensitivity, genotyping and quantitative accuracy. This article shows a overview of the application of real-time quantitative PCR technique to detecte group A rotavirus.

  6. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

    PubMed Central

    Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-01-01

    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

  7. Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma

    PubMed Central

    2010-01-01

    Background The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients. PMID:21040522

  8. Real-time image subtraction using phase reversal technique

    NASA Astrophysics Data System (ADS)

    Venkateswara Rao, Vuyyuru; Krishna Mohan, Nandigana K.

    1999-10-01

    A simple coherent interferometric processing method for image subtraction in real-time is presented. The proposed method is based on interferometric principle using Mach- Zehnder interferometer. The phase reversal is accomplished by varying the pressure within an air-filled quartz cell inserted in one of the arms of the interferometer. Initially, the interferometer is aligned to obtain broad interference fringes in the cell region. Then the input imageries are introduced in both the arms of the interferometer and adjusted for exact registration as seen in the plane of observation. By introducing a phase change of (pi) -rad between the two arms of the interferometer, the difference between the inputs is detected in real-time on the monitor. Phase shift calibration and information processing of the proposed method is presented with the results.

  9. Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts.

    PubMed

    Coates, Dawn E; Zafar, Sobia; Milne, Trudy J

    2017-01-01

    The use of quantitative real-time reverse transcriptase PCR (qRT(2)-PCR) for the identification of differentially regulated genes is a powerful technology. The protocol presented here uses qRT(2)-PCR gene arrays to investigate the regulation of 84 angiogenic related genes in human primary alveolar osteoblasts following treatment with the bisphosphonate, zoledronic acid (ZA), and geranylgeraniol (GGOH). GGOH has potential as a therapeutic agent for Bisphosphate-Related Osteonecrosis of the Jaw (BRONJ), a serious side-effect resulting from the treatment for metastatic cancer (Zafar et al., J Oral Pathol Med 43:711-721, 2014; Ruggiero, Ann NY Acad Sci 1218:38-46, 2011). The isolation of the primary osteoblast cells follows the methods previously described (Dillon et al., Methods Mol Biol 816:3-18, 2012) with a new RNA extraction technique described fully. The method highlights the importance of obtaining high-quality RNA which is DNA-free. Relative levels of gene expression are normalized against selected housekeeping genes (HKG) and a number of examples of how fold regulation (2(-∆∆Cq)) and gene expression level (2(-∆Cq)) data can be presented are given.

  10. Citrus stubborn disease incidence determined by quantitative real time PCR

    USDA-ARS?s Scientific Manuscript database

    Quantitative real-time (q) PCR was developed for detection of Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), using the DNA binding fluorophore SYBR Green I. The primer pair, P58-3f/4r, developed based on sequences from the P58 putative adhesin multigene of the pathogen result...

  11. Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

    PubMed

    Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

    2017-04-01

    Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10(2) to 10(3) copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  12. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    PubMed Central

    Langel, Ülo

    2016-01-01

    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies. PMID:27101307

  13. High-throughput quantitative real-time PCR.

    PubMed

    Arany, Zoltan P

    2008-07-01

    Recent technical advances in quantitative real-time PCR (qRT-PCR) have allowed for extensive miniaturization, thereby rendering the technique amenable to high-throughput assays. Large numbers of different nucleic acids can now rapidly be measured quantitatively. Many investigations can benefit from this approach, including determination of gene expression in hundreds of samples, determination of hundreds of genes in a few samples, or even quantification of nucleic acids other than mRNA. A simple technique is described here to quantify 1880 transcripts of choice from any number of starting RNA samples.

  14. A microarray scanner for the real-time quantitative detection

    NASA Astrophysics Data System (ADS)

    Liu, Quanjun; Zhuang, Ying; Wu, Lingwei; Wu, Zhongwei; Hu, Song; Lu, Zuhong

    2007-05-01

    The real-time and quantitative detection assay is important for the gene detection. With the TaqMan probes for the detection based polymerase chain reaction (PCR), four targets could be checked in a single process in solution assay. A new method is developed to immobilize the TaqMan probes on a microarray, which could be used to the multi-target gene fragment quantitative detection with PCR. A new type microarray scanner is designed for the assay. A thermocycler system was built into the scanner platform. A new type of the vessel sealed with the gene amplification solution which could perform the thermo-cycling and scanning. To decrease the background intensity a confocal system was used as the fluorescent intensity detection in the scanner. To calculate the gene quantity, a standard liner graph was draw with the fluorescent intensity versus the cycles. From the standard liner, the quantity of the original gene fragment could be calculated in time with the cycles. This scanner offers the great advantage of real-time quantitative detection of DNA targets in a microarray.

  15. Quantitative analysis of influenza A (H3N2) E119V and R292K variants in clinical specimens by real-time reverse transcription polymerase chain reaction.

    PubMed

    Sato, Masatoki; Honzumi, Ken; Sato, Toshiko; Hashimoto, Koichi; Watanabe, Masahiro; Miyazaki, Kyohei; Kawasaki, Yukihiko; Hosoya, Mitsuaki

    2015-07-01

    Because influenza virus isolates after cell culture are required to determine their susceptibility to neuraminidase inhibitors, the differences in normal or low-susceptibility variant population frequencies between clinical samples and isolates have not been considered. To identify variations in low-susceptibility populations in clinical samples after initiation of oseltamivir and zanamivir therapy by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). We measured the populations of the low-susceptibility influenza A H3N2 variants E119V and R292K by qRT-PCR using 305 nasal aspiration samples collected over time from 13, 16, and 11 patients treated with no neuraminidase inhibitors, oseltamivir, and zanamivir, respectively. The variant population in the isolates was also determined when the population of low-susceptibility variants in the clinical samples increased following treatment. Moreover, the susceptibility of all isolates was measured. The E119V variant was detected in only one patient during oseltamivir therapy, exhibiting decreased susceptibility to oseltamivir. Prior to treatment, R292K variants were detected in all clinical samples; however, they comprised only a small fraction of the total population. The proportion of the R292K variant in clinical samples increased for 6/27 (22.2%) patients treated with oseltamivir or zanamivir, whereas an increase in the proportion of the R292K variant in virus isolates was observed in only one patient. Discrepancies in the proportion of R292K variants between clinical samples and isolates should be suspected in clinical settings. qRT-PCR is useful for quantitative analysis of drug-resistant influenza virus and for immediate notification of the result. Copyright © 2015. Published by Elsevier B.V.

  16. Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

    PubMed

    Hindiyeh, M Y; Moran-Gilad, J; Manor, Y; Ram, D; Shulman, L M; Sofer, D; Mendelson, E

    2014-02-20

    In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay’s performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

  17. Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

    PubMed

    Zhao, Jing; Zhao, Jin-Yin; Chen, Zhi-Xia; Zhong, Wei; Li, Long-Yun; Liu, Li-Cheng; Hu, Xiao-Xu; Chen, Wei-Jun; Wang, Meng-Zhao

    2016-12-20

    Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

  18. Diagnosis of aerobic vaginitis by quantitative real-time PCR.

    PubMed

    Rumyantseva, T A; Bellen, G; Savochkina, Y A; Guschin, A E; Donders, G G G

    2016-07-01

    To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. Some level of AV was diagnosed in 23 (23.7 %) cases. Various concentrations of Enterobacteriacea, Staphylococcus spp., Streptococcus spp. were detected an all patients. Enterococcus spp. were detected in 76 (78.3 %) cases. Summarized concentrations of aerobes were tenfold higher in AV-positive compared to AV-negative cases [7.30lg vs 6.06lg (p = 0.02)]. Concentrations of aerobes in severe, moderate and light AV cases did not vary significantly (p = 0.14). Concentration of lactobacilli was 1000-fold lower in AV-positive cases compared to normal cases (5.3lg vs 8.3lg, p < 0.0001). Streptococcus spp. dominated in the majority of AV-positive cases [19/22 (86.4 %) samples]. The relation of high loads of aerobes to the low numbers of Lactobacilli are a reliable marker for the presence of AV and could substitute microscopy as a test. PCR may be a good standardized substitution for AV diagnosis in settings where well-trained microscopists are lacking.

  19. Real-time quantitative phase imaging for cell studies

    NASA Astrophysics Data System (ADS)

    Pham, Hoa Vinh

    Most biological cells are not clearly visible with a bright field microscope. Several methods have been developed to improve contrast in cell imaging, including use of exogenous contrast agents such as fluorescence microscopy, as well as utilizing properties of light-specimen interaction for optics design, to reveal the endogenous contrast, such as phase contrast microscopy (PCM) and differential interference contrast (DIC) microscopy. Although PCM and DIC methods significantly improve the image contrast without the need for staining agents, they only provide qualitative information about the phase change induced by the cells as light passes through them. Quantitative phase imaging (QPI) has recently emerged as an effective imaging tool which provides not only better image contrast but also cell-induced phase shifts in the optical pathlength, thus allowing nanometer-scale measurements of structures and dynamics of the cells. Other important aspects of an imaging system are its imaging speed and throughput. High-throughput, high-speed, real-time quantitative phase imaging with high spatial and temporal sensitivity is highly desirable in many applications including applied physics and biomedicine. In this dissertation, to address this need, I discuss the development of such an imaging system that includes the white light diffraction phase microscopy (wDPM), a new optical imaging method, and image reconstruction/analysis algorithms using graphics processing units (GPUs). wDPM can measure optical pathlength changes at nanometer scale both spatially and temporally with single-shot image acquisition, enabling very fast imaging. I also exploit the broadband spectrum of white light used as the light source in wDPM to develop a system called spectroscopic diffraction phase microscopy (sDPM). This sDPM system allows QPI measurements at several wavelengths, which solves the problem of thickness and refractive index coupling in the phase shifts induced by the cell, and which

  20. Quantitative real-time single particle analysis of virions

    PubMed Central

    Heider, Susanne; Metzner, Christoph

    2014-01-01

    Providing information about single virus particles has for a long time been mainly the domain of electron microscopy. More recently, technologies have been developed—or adapted from other fields, such as nanotechnology—to allow for the real-time quantification of physical virion particles, while supplying additional information such as particle diameter concomitantly. These technologies have progressed to the stage of commercialization increasing the speed of viral titer measurements from hours to minutes, thus providing a significant advantage for many aspects of virology research and biotechnology applications. Additional advantages lie in the broad spectrum of virus species that may be measured and the possibility to determine the ratio of infectious to total particles. A series of disadvantages remain associated with these technologies, such as a low specificity for viral particles. In this review we will discuss these technologies by comparing four systems for real-time single virus particle analysis and quantification. PMID:24999044

  1. Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR.

    PubMed

    Schibler, Manuel; Yerly, Sabine; Vieille, Gaël; Docquier, Mylène; Turin, Lara; Kaiser, Laurent; Tapparel, Caroline

    2012-09-01

    Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.

  2. Quantitative real-time single particle analysis of virions

    SciTech Connect

    Heider, Susanne; Metzner, Christoph

    2014-08-15

    Providing information about single virus particles has for a long time been mainly the domain of electron microscopy. More recently, technologies have been developed—or adapted from other fields, such as nanotechnology—to allow for the real-time quantification of physical virion particles, while supplying additional information such as particle diameter concomitantly. These technologies have progressed to the stage of commercialization increasing the speed of viral titer measurements from hours to minutes, thus providing a significant advantage for many aspects of virology research and biotechnology applications. Additional advantages lie in the broad spectrum of virus species that may be measured and the possibility to determine the ratio of infectious to total particles. A series of disadvantages remain associated with these technologies, such as a low specificity for viral particles. In this review we will discuss these technologies by comparing four systems for real-time single virus particle analysis and quantification. - Highlights: • We introduce four methods for virus particle-based quantification of viruses. • They allow for quantification of a wide range of samples in under an hour time. • The additional measurement of size and zeta potential is possible for some.

  3. QUANTIFICATION OF ENTEROVIRUS AND HEPATITIS A VIRUSES IN WELLS AND SPRINGS IN EAST TENNESSEE USING REAL-TIME REVERSE TRANSCIPTION PCR

    EPA Science Inventory

    This project involves development, validation testing and application of a fast, efficient method of quantitatively measuring occurrence and concentration of common human viral pathogens, enterovirus and hepatitis A virus, in ground water samples using real-time reverse transcrip...

  4. QUANTIFICATION OF ENTEROVIRUS AND HEPATITIS A VIRUSES IN WELLS AND SPRINGS IN EAST TENNESSEE USING REAL-TIME REVERSE TRANSCIPTION PCR

    EPA Science Inventory

    This project involves development, validation testing and application of a fast, efficient method of quantitatively measuring occurrence and concentration of common human viral pathogens, enterovirus and hepatitis A virus, in ground water samples using real-time reverse transcrip...

  5. Real-time quantitative PCR assay for monitoring of nervous necrosis virus infection in grouper aquaculture.

    PubMed

    Kuo, Hsiao-Che; Wang, Ting-Yu; Chen, Peng-Peng; Chen, Young-Mao; Chuang, Hui-Ching; Chen, Tzong-Yueh

    2011-03-01

    Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.

  6. Automated quantitation of tissue components using real-time spectroscopy

    NASA Astrophysics Data System (ADS)

    Benaron, David A.; Rubinski, Boris; Hintz, Susan R.; Duckworth, Joshua L.; Murphy, Aileen L.; Price, John W., Jr.; Liu, Frank W.; Otten, David M.; Stevenson, David K.; Cheong, Wai-Fung; Kermit, Eben L.

    1998-01-01

    Each tissue has a unique spectral signature (e.g. liver looks distinct from bowel due to differences in both absorbance and in the way the tissue scatters light). Therefore, we suspect that automated discrimination among tissue types (e.g. blood, nerve, artery, vein, muscle) or tissue state (frozen, unfrozen, viable, dead) is feasible. In this study, we investigated our ability to detect hidden structures (such as blood vessels) or events (such as tissue ablation via freezing) using optical systems. For blood vessel localization, a key step in vascular access, we resolved the component concentration of hemoglobin measured within the tissue, and found that blood vessel depth and direction could be determined. For freezing detection, we found that changes in effective absorbance during freezing allowed the freezing process to be monitored spectroscopically. Such optical techniques may usher in use of light-assisted medical diagnosis, leading to automated and portable diagnostic devices which enable real-time diagnostics and monitoring during medical interventions, such as cryoablation or vascular access.

  7. Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA▿ †

    PubMed Central

    González-Escalona, Narjol; Hammack, Thomas S.; Russell, Mindi; Jacobson, Andrew P.; De Jesús, Antonio J.; Brown, Eric W.; Lampel, Keith A.

    2009-01-01

    Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 103 CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 105 and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/∼ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers. PMID:19376910

  8. Performance of the RealStar Chikungunya Virus Real-Time Reverse Transcription-PCR Kit▿

    PubMed Central

    Panning, Marcus; Hess, Markus; Fischer, Waldemar; Grywna, Klaus; Pfeffer, Martin; Drosten, Christian

    2009-01-01

    A novel commercial Chikungunya virus real-time reverse transcription-PCR (RT-PCR) kit was evaluated on a comprehensive panel of original patient samples. The assay was 100% sensitive and specific in comparison to a published real-time RT-PCR. Viral loads from both assays were highly correlated. The kit proved to be suitable for routine use in patient care. PMID:19625474

  9. Real Time Quantitative Radiological Monitoring Equipment for Environmental Assessment

    SciTech Connect

    John R. Giles; Lyle G. Roybal; Michael V. Carpenter

    2006-03-01

    and measures. These analyses are combined to provide real-time areal activity and coverage maps that are displayed to the operator as the survey progresses. The flexible functionality of the INL systems are well suited to multiple roles supporting homeland security needs.

  10. Real Time Quantitative 3-D Imaging of Diffusion Flame Species

    NASA Technical Reports Server (NTRS)

    Kane, Daniel J.; Silver, Joel A.

    1997-01-01

    A low-gravity environment, in space or ground-based facilities such as drop towers, provides a unique setting for study of combustion mechanisms. Understanding the physical phenomena controlling the ignition and spread of flames in microgravity has importance for space safety as well as better characterization of dynamical and chemical combustion processes which are normally masked by buoyancy and other gravity-related effects. Even the use of so-called 'limiting cases' or the construction of 1-D or 2-D models and experiments fail to make the analysis of combustion simultaneously simple and accurate. Ideally, to bridge the gap between chemistry and fluid mechanics in microgravity combustion, species concentrations and temperature profiles are needed throughout the flame. However, restrictions associated with performing measurements in reduced gravity, especially size and weight considerations, have generally limited microgravity combustion studies to the capture of flame emissions on film or video laser Schlieren imaging and (intrusive) temperature measurements using thermocouples. Given the development of detailed theoretical models, more sophisticated studies are needed to provide the kind of quantitative data necessary to characterize the properties of microgravity combustion processes as well as provide accurate feedback to improve the predictive capabilities of the computational models. While there have been a myriad of fluid mechanical visualization studies in microgravity combustion, little experimental work has been completed to obtain reactant and product concentrations within a microgravity flame. This is largely due to the fact that traditional sampling methods (quenching microprobes using GC and/or mass spec analysis) are too heavy, slow, and cumbersome for microgravity experiments. Non-intrusive optical spectroscopic techniques have - up until now - also required excessively bulky, power hungry equipment. However, with the advent of near-IR diode

  11. Real Time Quantitative 3-D Imaging of Diffusion Flame Species

    NASA Technical Reports Server (NTRS)

    Kane, Daniel J.; Silver, Joel A.

    1997-01-01

    A low-gravity environment, in space or ground-based facilities such as drop towers, provides a unique setting for study of combustion mechanisms. Understanding the physical phenomena controlling the ignition and spread of flames in microgravity has importance for space safety as well as better characterization of dynamical and chemical combustion processes which are normally masked by buoyancy and other gravity-related effects. Even the use of so-called 'limiting cases' or the construction of 1-D or 2-D models and experiments fail to make the analysis of combustion simultaneously simple and accurate. Ideally, to bridge the gap between chemistry and fluid mechanics in microgravity combustion, species concentrations and temperature profiles are needed throughout the flame. However, restrictions associated with performing measurements in reduced gravity, especially size and weight considerations, have generally limited microgravity combustion studies to the capture of flame emissions on film or video laser Schlieren imaging and (intrusive) temperature measurements using thermocouples. Given the development of detailed theoretical models, more sophisticated studies are needed to provide the kind of quantitative data necessary to characterize the properties of microgravity combustion processes as well as provide accurate feedback to improve the predictive capabilities of the computational models. While there have been a myriad of fluid mechanical visualization studies in microgravity combustion, little experimental work has been completed to obtain reactant and product concentrations within a microgravity flame. This is largely due to the fact that traditional sampling methods (quenching microprobes using GC and/or mass spec analysis) are too heavy, slow, and cumbersome for microgravity experiments. Non-intrusive optical spectroscopic techniques have - up until now - also required excessively bulky, power hungry equipment. However, with the advent of near-IR diode

  12. Quantitative Real-Time PCR Fecal Source Identification in the ...

    EPA Pesticide Factsheets

    Rivers in the Tillamook Basin play a vital role in supporting a thriving dairy and cheese-making industry, as well as providing a safe water resource for local human and wildlife populations. Historical concentrations of fecal bacteria in these waters are at times too high to allow for safe use leading to economic loss, endangerment of local wildlife, and poor conditions for recreational use. In this study, we employ host-associated qPCR methods for human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution combined with high-resolution geographic information system (GIS) land use data and general indicator bacteria measurements to elucidatewater quality spatial and temporal trends. Water samples (n=584) were collected over a 1-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries (Tillamook Basin, OR). A total of 16.6% of samples (n=97) yielded E. coli levels considered impaired based on Oregon Department of Environmental Quality bacteria criteria (406 MPN/100mL). Hostassociated genetic indicators were detected at frequencies of 39.2% (HF183/BacR287), 16.3% (HumM2), 74.6% (Rum2Bac), 13.0% (CowM2), 26.7% (CowM3), 19.8% (DG3), 3.2% (DG37), and 53.4% (GFD) across all water samples (n=584). Seasonal trends in avian, cattle, and human fecal pollution sources were evident over the study area. On a sample site basis, quantitative fecal source identification and

  13. The quantitative real-time polymerase chain reaction for the analysis of plant gene expression.

    PubMed

    Fitzgerald, Timothy L; McQualter, Richard B

    2014-01-01

    The quantitative real-time polymerase chain reaction is used to simultaneously amplify and quantify a targeted DNA molecule. It can be used to determine exact copy number of a molecule within a sample and/or to compare the quantity of a molecule between samples. When combined with reverse transcription, it is a powerful tool for the analysis of gene expression, and it is widely used for this purpose in plant species. Here we provide an introduction to fundamental concepts relevant for the analysis of gene expression in plants using this technique and a protocol for quantification of the relative expression of a sucrose phosphate synthase gene along the maturation gradient of a sugarcane leaf.

  14. The workflow of single-cell expression profiling using quantitative real-time PCR

    PubMed Central

    Ståhlberg, Anders; Kubista, Mikael

    2014-01-01

    Biological material is heterogeneous and when exposed to stimuli the various cells present respond differently. Much of the complexity can be eliminated by disintegrating the sample, studying the cells one by one. Single-cell profiling reveals responses that go unnoticed when classical samples are studied. New cell types and cell subtypes may be found and relevant pathways and expression networks can be identified. The most powerful technique for single-cell expression profiling is currently quantitative reverse transcription real-time PCR (RT-qPCR). A robust RT-qPCR workflow for highly sensitive and specific measurements in high-throughput and a reasonable degree of multiplexing has been developed for targeting mRNAs, but also microRNAs, non-coding RNAs and most recently also proteins. We review the current state of the art of single-cell expression profiling and present also the improvements and developments expected in the next 5 years. PMID:24649819

  15. [Analysis of appropriate amount of template DNA for sinofiler kit by real time quantitative PCR technique].

    PubMed

    Li, Cheng-Tao; Guo, Hong; Lin, Yuan; Liu, Yan; Que, Ting-Zhi; Li, Li

    2008-04-01

    To explore the appropriate amount of template DNA for Sinofiler Kit. The DNA samples with ideally genotyped results by Sinofiler Kit were detected by real-time quantitative PCR assay. It was shown that 1.29-1.51 ng of template DNA in 12.5 microL reaction volume was optimal for STR genotyping with Sinofiler Kit. Real time quantitative PCR is an accurate and necessary technique for detection of appropriate amount of template DNA for different kits.

  16. Using Quantitative Real-Time PCR to Detect MicroRNA Expression Profile During Embryonic Stem Cell Differentiation.

    PubMed

    Pan, Xiaoping; Murashov, Alexander K; Stellwag, Edmund J; Zhang, Baohong

    2017-01-01

    Quantitative real-time PCR (qRT-PCR) is a reliable method to determine and monitor microRNA (miRNA) expression profiles in different cells, tissues, and organisms. Although there are several different strategies in performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequencing and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of quantitative real-time PCR for determining miRNA expression profiles during mouse embryonic stem cell differentiation. In this method, a mature miRNA sequence is first reverse transcribed into a long cDNA with a 40-50 nt miRNA-specific stem-loop primer; then, a standard real-time PCR reaction is performed for determining miRNA expression using a forward miRNA-specific primer and a universal reverse primer.

  17. Real-time quantitative fluorescence imaging using a single snapshot optical properties technique for neurosurgical guidance

    NASA Astrophysics Data System (ADS)

    Valdes, Pablo A.; Angelo, Joseph; Gioux, Sylvain

    2015-03-01

    Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

  18. Gene expression analysis by quantitative real-time PCR for floral tissues.

    PubMed

    Bustamante, Mariana; Jin, Jian; Casagran, Oriol; Nolan, Tania; Riechmann, José Luis

    2014-01-01

    Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler(®) 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment.

  19. A quantitative real-time PCR method for monitoring Clostridium botulinum type A in rice samples.

    PubMed

    Takahashi, Hajime; Takakura, Chikako; Kimura, Bon

    2010-04-01

    A quantitative real-time PCR using SYBR Green dye was developed to target the neurotoxin type A (boNT/A) gene of Clostridium botulinum type A. Primer specificity was confirmed by analyzing 63 strains including 5 strains of C. botulinum type A and 11 of non-type A C. botulinum. The highly similar amplification efficiencies of the real-time PCR assay were observed for 5 strains of C. botulinum type A. The DNA extraction with NucliSENS miniMAG provided sufficient performance to obtain the purified DNA from steamed rice samples and to develop the standard curve for the enumeration of C. botulinum in steamed rice samples. The real-time PCR assay could detect 10 cells per milliliter of 10 x rice homogenate, thus indicating that more than 100 C. botulinum cells per g of rice sample was quantifiable by the real-time PCR assay. The inoculation of aseptic rice samples with low numbers of C. botulinum type A cells revealed that the fate of inoculated C. botulinum type A cells in rice samples could be monitored accurately by the real-time PCR assay. These results indicate that the real-time PCR assay developed in this study provides rapid, effective, and quantitative monitoring of C. botulinum in steamed rice samples.

  20. Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV).

    PubMed

    Cook, R Frank; Cook, S J; Li, F Li; Montelaro, R C; Issel, C J

    2002-08-01

    A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6), 10(4) and 10(2) molecules.

  1. Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus

    PubMed Central

    Gu, Z.; Ingersoll, J.; Abdul-Ali, D.; Shi, L.; Pounds, S.; Caliendo, A. M.

    2013-01-01

    Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log10 versus 4 log10 copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R2 ≥ 0.98 in each of 6 regression models) and clinical samples (R2 = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR. PMID:23224089

  2. [Application of fluorescent real-time reverse transcriptase-polymerase chain reaction in detecting influenza viruses].

    PubMed

    Cheng, Xiao-wen; Zhou, Li; Zhao, Jin; Fang, Shi-song; Yu, Lei; Ye, Bao-ying; He, Jian-fan; Lu, Xing; Zhang, Zai-qing; Yang, Hong

    2004-09-01

    To apply fluorescent real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in detecting influenza viruses. A total of 207 oral swab samples were obtained in 16 collections from SARS patients and suspected influenza outbreak cases. They were subjected to influenza virus detection by fluorescent real-time RT-PCR, MDCK cell culture, and hemagglutinin inhibition assay. Out of 207 samples, 79 (38.16%) were positive for influenza viruses when tested by fluorescent real-time PCR, and 62 (29.95%) positive when tested by MDCK cell culture. There was a statistically significant difference between them (chi square=8.64, P less than 0.005). From 104 cases in 9 collections dual serum samples were obtainable. When tested with hemagglutinin inhibition assay, 64 cases (61.54%) showed a 4-fold increase against H3N2 antigen. This study showed that fluorescent real-time PCR is a reliable, sensitive, and fast method for detecting influenza viruses.

  3. Processing of gene expression data generated by quantitative real-time RT-PCR.

    PubMed

    Muller, Patrick Y; Janovjak, Harald; Miserez, André R; Dobbie, Zuzana

    2002-06-01

    Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.

  4. Reference gene selection for quantitative real-time PCR normalization in Quercus suber.

    PubMed

    Marum, Liliana; Miguel, Andreia; Ricardo, Cândido P; Miguel, Célia

    2012-01-01

    The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  5. Design of primers and probes for quantitative real-time PCR methods.

    PubMed

    Rodríguez, Alicia; Rodríguez, Mar; Córdoba, Juan J; Andrade, María J

    2015-01-01

    Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.

  6. [Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm, Helicoverna armigera].

    PubMed

    Chandra, G Sharath; Asokan, R; Manamohan, M; Kumar, N K K; Sita, T

    2014-01-01

    Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi), is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. β-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin, expression. These results facilitate accurate quantification of gene expression in H. armigera.

  7. Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

    EPA Science Inventory

    A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

  8. Quantitation of Bacillus clausii in biological samples by real-time polymerase chain reaction.

    PubMed

    Perotti, Mario; Mancini, Nicasio; Cavallero, Annalisa; Carletti, Silvia; Canducci, Filippo; Burioni, Roberto; Clementi, Massimo

    2006-06-01

    A real-time PCR assay targeting the highly specific erm34 sequence of Bacillus clausii DNA was developed and optimized. The quantitative assay showed a sensitivity level of 10(2) CFU/microl of sample. The method may represent a useful tool for monitoring the role of B. clausii as probiotic in vivo.

  9. Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

    EPA Science Inventory

    A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

  10. Evaluation of Quantitative Real-Time PCR Assays for Detection of Citrus Greening

    USDA-ARS?s Scientific Manuscript database

    Citrus huanglongbing (HLB), or citrus greening, is a serious and industry-limiting disease. Preliminary diagnoses can be made through visual symptoms, and greater certainty can be achieved through quantitative real-time PCR (qPCR). Several qPCR procedures are available including those by designed by...

  11. Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration es...

  12. Off-axis quantitative phase imaging processing using CUDA: toward real-time applications.

    PubMed

    Pham, Hoa; Ding, Huafeng; Sobh, Nahil; Do, Minh; Patel, Sanjay; Popescu, Gabriel

    2011-07-01

    We demonstrate real time off-axis Quantitative Phase Imaging (QPI) using a phase reconstruction algorithm based on NVIDIA's CUDA programming model. The phase unwrapping component is based on Goldstein's algorithm. By mapping the process of extracting phase information and unwrapping to GPU, we are able to speed up the whole procedure by more than 18.8× with respect to CPU processing and ultimately achieve video rate for mega-pixel images. Our CUDA implementation also supports processing of multiple images simultaneously. This enables our imaging system to support high speed, high throughput, and real-time image acquisition and visualization.

  13. Off-axis quantitative phase imaging processing using CUDA: toward real-time applications

    PubMed Central

    Pham, Hoa; Ding, Huafeng; Sobh, Nahil; Do, Minh; Patel, Sanjay; Popescu, Gabriel

    2011-01-01

    We demonstrate real time off-axis Quantitative Phase Imaging (QPI) using a phase reconstruction algorithm based on NVIDIA’s CUDA programming model. The phase unwrapping component is based on Goldstein’s algorithm. By mapping the process of extracting phase information and unwrapping to GPU, we are able to speed up the whole procedure by more than 18.8× with respect to CPU processing and ultimately achieve video rate for mega-pixel images. Our CUDA implementation also supports processing of multiple images simultaneously. This enables our imaging system to support high speed, high throughput, and real-time image acquisition and visualization. PMID:21750757

  14. Detection and quantitation of HPV DNA replication by Southern blotting and real-time PCR.

    PubMed

    Morgan, Iain M; Taylor, Ewan R

    2005-01-01

    This provides a brief introduction into the mechanism of DNA replication by the E1 and E2 proteins and describes the traditional Southern blotting technique that is used to monitor E1- and E2-mediated DNA replication. It also includes a novel real-time polymerase chain reaction (PCR) approach for monitoring E1- and E2-mediated DNA replication that has enhanced sensitivity and quantitation compared with Southern blotting, and a discussion of when to use the Southern blotting and real-time PCR techniques.

  15. Quantitative detection of residual porcine host cell DNA by real-time PCR.

    PubMed

    Chang, Jen-Ting; Chen, Yu-Chen; Chou, Yu-Chi; Wang, Shih-Rong

    2014-03-01

    All biological products are derived from complex living systems and are often mixed with large numbers of impurities. For reasons of safety, residual host-cell DNA must be eliminated during processing. To assay host-cell DNA content in biopharmaceutical products derived from porcine sources, this study applies the quantitative real-time polymerase chain reaction (Q-PCR) method. The optimized assay in this study is based on the pol region of the porcine endogenous retrovirus (PERV). Assay validation results demonstrate that the proposed assay has appropriate accuracy, preciseness, reproducibility, and sensitivity. Primer and probe specificity are evaluated in real-time Q-PCR reactions using genomic DNA from rabbit, mouse, cat, hamster, monkey, human cell, yeast, and Escherichia coli as templates. The sensitivity of real-time Q-PCR is determined using genomic DNA from the porcine kidney cell line. The reliable detection range is within 0.5-10(5) pg/reaction. The limit of quantitation is 500 fg. The sensitivity of the assay meets the authority criterion. Moreover, the assay is applied to determine the level of host-cell DNA in recombinant human coagulation factor IX (rhFIX) from transgenic pigs. The real-time Q-PCR assay is thus a promising new tool for quantitative detection and clearance validation of residual porcine DNA when manufacturing recombinant therapeutics.

  16. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    PubMed

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-05

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.

    PubMed

    Ploemen, Ivo H J; Prudêncio, Miguel; Douradinha, Bruno G; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J F; Hermsen, Cornelus C; Sauerwein, Robert W; Baptista, Fernanda G; Mota, Maria M; Waters, Andrew P; Que, Ivo; Lowik, Clemens W G M; Khan, Shahid M; Janse, Chris J; Franke-Fayard, Blandine M D

    2009-11-18

    The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of

  18. Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

    PubMed Central

    Douradinha, Bruno G.; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J. F.; Hermsen, Cornelus C.; Sauerwein, Robert W.; Baptista, Fernanda G.; Mota, Maria M.; Waters, Andrew P.; Que, Ivo; Lowik, Clemens W. G. M.; Khan, Shahid M.; Janse, Chris J.; Franke-Fayard, Blandine M. D.

    2009-01-01

    The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of

  19. Real-time duplex PCR for simultaneous HPV 16 and HPV 18 DNA quantitation.

    PubMed

    Jacquin, Elise; Saunier, Maëlle; Mauny, Frédéric; Schwarz, Elisabeth; Mougin, Christiane; Prétet, Jean-Luc

    2013-11-01

    HPV 16 and HPV 18 are responsible for more than 75% of cervical cancers and high HPV 16 loads are associated with both prevalent and incident lesions. The objective of the present study was to develop a method allowing the detection and quantitation of HPV 16 and 18 DNA to improve future strategies for cervical cancer screening. A duplex real-time PCR allowing the simultaneous quantitation of both HPV 16 and HPV 18 was carried out. Mixes of HPV 16 and HPV 18 whole genome plasmids were prepared to test a wide range of viral DNA concentrations. The values obtained for each mix of plasmids with the simplex and the duplex PCR were very close to the theoretical values except when a HPV type represented only 1:1000 genome equivalent or lower than the concurrent type. Cervical samples harboring HPV 16, HPV 18 or both types were tested by comparing the results with simplex and duplex real-time PCR assays. HPV 16 and HPV 18 genome titers were similar with the two assays. In conclusion, the real-time duplex PCR proved to be robust for HPV 16 and HPV 18 DNA quantitation.

  20. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    PubMed

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  1. Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

    PubMed

    Nguyen, T L; Lim, Y J; Kim, D-H; Austin, B

    2016-01-01

    Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples. © 2014 John Wiley & Sons Ltd.

  2. A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

    PubMed Central

    Tolardo, Aline Lavado; de Souza, William Marciel; Romeiro, Marilia Farignoli; Vieira, Luiz Carlos; Luna, Luciano Kleber de Souza; Henriques, Dyana Alves; de Araujo, Jansen; Siqueira, Carlos Eduardo Hassegawa; Colombo, Tatiana Elias; Aquino, Victor Hugo; da Fonseca, Benedito Antonio Lopes; Bronzoni, Roberta Vieira de Morais; Nogueira, Maurício Lacerda; Durigon, Edison Luiz; Figueiredo, Luiz Tadeu Moraes

    2016-01-01

    Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections. PMID:27276185

  3. Legionellosis and Lung Abscesses: Contribution of Legionella Quantitative Real-Time PCR to an Adapted Followup

    PubMed Central

    Descours, G.; Tellini, C.; Flamens, C.; Philit, F.; Celard, M.; Etienne, J.; Lina, G.; Jarraud, S.

    2013-01-01

    We report a case of severe Legionnaires' disease (LD) complicated by a lung abscess in an immunocompetent patient who required ECMO therapy and thoracic surgery. The results of repeated Legionella quantitative real-time PCR performed on both sera and respiratory samples correlated with the LD severity and the poor clinical outcome. Moreover, the PCR allowed for the detection of Legionella DNA in the lung abscess specimen, which was negative when cultured for Legionella. This case report provides a logical basis for further investigations to examine whether the Legionella quantitative PCR could improve the assessment of LD severity and constitute a prognostic marker. PMID:23862082

  4. Real-time quantitative phase reconstruction in off-axis digital holography using multiplexing.

    PubMed

    Girshovitz, Pinhas; Shaked, Natan T

    2014-04-15

    We present a new approach for obtaining significant speedup in the digital processing of extracting unwrapped phase profiles from off-axis digital holograms. The new technique digitally multiplexes two orthogonal off-axis holograms, where the digital reconstruction, including spatial filtering and two-dimensional phase unwrapping on a decreased number of pixels, can be performed on both holograms together, without redundant operations. Using this technique, we were able to reconstruct, for the first time to our knowledge, unwrapped phase profiles from off-axis holograms with 1 megapixel in more than 30 frames per second using a standard single-core personal computer on a MATLAB platform, without using graphic-processing-unit programming or parallel computing. This new technique is important for real-time quantitative visualization and measurements of highly dynamic samples and is applicable for a wide range of applications, including rapid biological cell imaging and real-time nondestructive testing. After comparing the speedups obtained by the new technique for holograms of various sizes, we present experimental results of real-time quantitative phase visualization of cells flowing rapidly through a microchannel.

  5. Development of Multiplexed Real-Time Quantitative PCR Assay for Detecting Human Adenoviruses

    PubMed Central

    Huang, Meei-Li; Nguy, Long; Ferrenberg, James; Boeckh, Michael; Cent, Anne; Corey, Lawrence

    2008-01-01

    Adenoviruses (AdV) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Due to the large number of existing Adv types, few real-time quantitative AdV PCR assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into two separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (Types 1-49) available from ATCC. We then subsequently multiplexed all the primers and probes into one reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/ml plasma). In a retrospective evaluation we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplant (HSCT) between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for adenovirus testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real time PCR assay allows rapid, sensitive and specific quantification of all currently defined adenoviruses into either two or one multiplex assay for clinical samples. PMID:18707838

  6. Quantitation of HIV-1 RNA in breast milk by real time PCR.

    PubMed

    Becquart, Pierre; Foulongne, Vincent; Willumsen, Juana; Rouzioux, Christine; Segondy, Michel; Van de Perre, Philippe

    2006-04-01

    HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

  7. Quantitative real-time monitoring of dryer effluent using fiber optic near-infrared spectroscopy.

    PubMed

    Harris, S C; Walker, D S

    2000-09-01

    This paper describes a method for real-time quantitation of the solvents evaporating from a dryer. The vapor stream in the vacuum line of a dryer was monitored in real time using a fiber optic-coupled acousto-optic tunable filter near-infrared (AOTF-NIR) spectrometer. A balance was placed in the dryer, and mass readings were recorded for every scan of the AOTF-NIR. A partial least-squares (PLS) calibration was subsequently built based on change in mass over change in time for solvents typically used in a chemical manufacturing plant. Controlling software for the AOTF-NIR was developed. The software collects spectra, builds the PLS calibration model, and continuously fits subsequently collected spectra to the calibration, allowing the operator to follow the mass loss of solvent from the dryer. The results indicate that solvent loss can be monitored and quantitated in real time using NIR for the optimization of drying times. These time-based mass loss values have also been used to calculate "dynamic" vapor density values for the solvents. The values calculated are in agreement with values determined from the ideal gas law and could prove valuable as tools to measure temperature or pressure indirectly.

  8. Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    PubMed

    Oguchi, Taichi; Onishi, Mari; Minegishi, Yasutaka; Kurosawa, Yasunori; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2009-06-01

    A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

  9. Fast processing of quantitative phase profiles from off-axis interferograms for real-time applications

    NASA Astrophysics Data System (ADS)

    Girshovitz, Pinhas; Shaked, Natan T.

    2015-03-01

    We review new and efficient algorithms, lately presented by us, for rapid reconstruction of quantitative phase maps from off-axis digital interferograms. These algorithms improve the conventional Fourier-based algorithm by using the Fourier transforms and the phase unwrapping process more efficiently, and thus decrease the calculation complexity required for extracting the sample phase map from the recorded interferograms. Using the new algorithms, on a standard personal computer without using the graphic processing-unit programming or parallel computing, we were able to speed up the processing and reach frame rates of up to 45 frames per second for one megapixel off-axis interferograms. These capabilities allow real-time visualization, calculation and data extraction for dynamic samples and processes, inspected by off-axis digital holography. Specific applications include biological cell imaging without labeling and real-time nondestructive testing.

  10. Real-Time Quantitative PCR Assay for Monitoring of Nervous Necrosis Virus Infection in Grouper Aquaculture▿†

    PubMed Central

    Kuo, Hsiao-Che; Wang, Ting-Yu; Chen, Peng-Peng; Chen, Young-Mao; Chuang, Hui-Ching; Chen, Tzong-Yueh

    2011-01-01

    Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r2 = 0.99) between threshold cycle (CT) and RNA quantities, which allowed identification of infected groupers by the CT value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture. PMID:21233077

  11. Quantitative gene expression profiles in real time from expressed sequence tag databases.

    PubMed

    Funari, Vincent A; Voevodski, Konstantin; Leyfer, Dimitry; Yerkes, Laura; Cramer, Donald; Tolan, Dean R

    2010-01-01

    An accumulation of expressed sequence tag (EST) data in the public domain and the availability of bioinformatic programs have made EST gene expression profiling a common practice. However, the utility and validity of using EST databases (e.g., dbEST) has been criticized, particularly for quantitative assessment of gene expression. Problems with EST sequencing errors, library construction, EST annotation, and multiple paralogs make generation of specific and sensitive qualitative arid quantitative expression profiles a concern. In addition, most EST-derived expression data exists in previously assembled databases. The Virtual Northern Blot (VNB) (http: //tlab.bu.edu/vnb.html) allows generation, evaluation, and optimization of expression profiles in real time, which is especially important for alternatively spliced, novel, or poorly characterized genes. Representative gene families with variable nucleotide sequence identity, tissue specificity, and levels of expression (bcl-xl, aldoA, and cyp2d9) are used to assess the quality of VNB's output. The profiles generated by VNB are more sensitive and specific than those constructed with ESTs listed in preindexed databases at UCSC and NCBI. Moreover, quantitative expression profiles produced by VNB are comparable to quantization obtained from Northern blots and qPCR. The VNB pipeline generates real-time gene expression profiles for single-gene queries that are both qualitatively and quantitatively reliable.

  12. Quantitative Gene Expression Profiles in Real Time From Expressed Sequence Tag Databases

    PubMed Central

    FUNARI, VINCENT A.; VOEVODSKI, KONSTANTIN; LEYFER, DIMITRY; YERKES, LAURA; CRAMER, DONALD; TOLAN, DEAN R.

    2010-01-01

    An accumulation of expressed sequence tag (EST) data in the public domain and the availability of bioinformatic programs have made EST gene expression profiling a common practice. However, the utility and validity of using EST databases (e.g., dbEST) has been criticized, particularly for quantitative assessment of gene expression. Problems with EST sequencing errors, library construction, EST annotation, and multiple paralogs make generation of specific and sensitive qualitative and quantitative expression profiles a concern. In addition, most EST-derived expression data exists in previously assembled databases. The Virtual Northern Blot (VNB) (http://tlab.bu.edu/vnb.html) allows generation, evaluation, and optimization of expression profiles in real time, which is especially important for alternatively spliced, novel, or poorly characterized genes. Representative gene families with variable nucleotide sequence identity, tissue specificity, and levels of expression (bcl-xl, aldoA, and cyp2d9) are used to assess the quality of VNB’s output. The profiles generated by VNB are more sensitive and specific than those constructed with ESTs listed in preindexed databases at UCSI and NCBI. Moreover, quantitative expression profiles produced by VNB are comparable to quantization obtained from Northern blots and qPCR. The VNB pipeline generates real-time gene expression profiles for single-gene queries that are both qualitatively and quantitatively reliable. PMID:20635574

  13. Real-time structured light-based otoscopy for quantitative measurement of eardrum deformation

    NASA Astrophysics Data System (ADS)

    Van der Jeught, Sam; Dirckx, Joris J. J.

    2017-01-01

    An otological profilometry device based on real-time structured light triangulation is presented. A clinical otoscope head is mounted onto a custom-handheld unit containing both a small digital light projector and a high-speed digital camera. Digital fringe patterns are projected onto the eardrum surface and are recorded at a rate of 120 unique frames per second. The relative angle between projection and camera axes causes the projected patterns to appear deformed by the eardrum shape, allowing its full-field three-dimensional (3-D) surface map to be reconstructed. By combining hardware triggering between projector and camera with a dedicated parallel processing pipeline, the proposed system is capable of acquiring a live stream of point clouds of over 300,000 data points per frame at a rate of 40 Hz. Real-time eardrum profilometry adds an additional dimension of depth to the standard two-dimensional otoscopy image and provides a noninvasive tool to enhance the qualitative depth perception of the clinical operator with quantitative 3-D data. Visualization of the eardrum from different perspectives can improve the diagnosis of existing and the detection of impending middle ear pathology. The capability of the device to detect small middle ear pressure changes by monitoring eardrum deformation in real time is demonstrated.

  14. New buffers to improve the quantitative real-time polymerase chain reaction.

    PubMed

    Ahmad, Ashraf; Ghasemi, Jahan

    2007-08-01

    Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.

  15. Multiplex real-time reverse transcription-PCR assay for determination of hepatitis C virus genotypes.

    PubMed

    Cook, Linda; Sullivan, KaWing; Krantz, Elizabeth M; Bagabag, Arthur; Jerome, Keith R

    2006-11-01

    A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.

  16. Quantitation of HIV-1 by real-time amplification and detection

    NASA Astrophysics Data System (ADS)

    Jung, Paul M.; Yang, Naiquan; Kroeger, Paul E.

    1998-04-01

    A model assay for HIV-1 using a non-competitive internal standard in quantitative RT-PCR was coupled with real-time detection of both analyte and internal standard (IS) signals in a closed system. Real-time detection by the PE-ABI Prism 7700 relied on TaqMan probes specific for HIV and IS. The exogenous, non-competitive IS RNA was added in the same, known amount to a series of HIV RNA standards. The threshold cycle ratio from this internal standard calibration curve was used in the quantitation of HIV. Two configurations of reporter labels were compared. The HEX-HIV:FAM-IS system was the most precise, with nearly half-log discrimination over a range of 102 through 105 copies HIV-1 RNA. The FAM- HIV:HEX-IS system was less precise, but more sensitive and resistant to sample inhibition. The analysis of these signals and their impact on the range and precision of HIV quantitation is discussed. The design and synthesis of the fluorescently-labelled probes is also described.

  17. Quantitative analysis of periodontal pathogens by ELISA and real-time polymerase chain reaction.

    PubMed

    Hamlet, Stephen M

    2010-01-01

    The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, these methods also underpin more recent epidemiological analyses of the impact of periodontal disease on general health. Given the complex milieu of over 700 species of microorganisms known to exist within the complex biofilms found in the oral cavity, the identification and enumeration of oral periodontopathogens has not been an easy task. In recent years however, some of the intrinsic limitations of the more traditional microbiological analyses previously used have been overcome with the advent of immunological and molecular analytical methods. Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications. Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons as they accumulate in "real time" allowing subsequent quantitation. These methods enable the accurate quantitation of as few as 10(2) (using rtPCR) to 10(4) (using ELISA) periodontopathogens in dental plaque samples.

  18. Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus

    PubMed Central

    Wang, Kai; Niu, Yi; Wang, Qijun; Liu, Haili; Jin, Yi

    2017-01-01

    Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to

  19. Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus.

    PubMed

    Wang, Kai; Niu, Yi; Wang, Qijun; Liu, Haili; Jin, Yi; Zhang, Shenglin

    2017-01-01

    Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to

  20. On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

    NASA Technical Reports Server (NTRS)

    Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

    2015-01-01

    NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of

  1. Evaluation of internal control for gene expression in Phalaenopsis by quantitative real-time PCR.

    PubMed

    Yuan, Xiu-Yun; Jiang, Su-Hua; Wang, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2014-07-01

    The selection of appropriate reference genes is one of the most important steps to obtain reliable results for normalizing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of MADS-box gene in Phalaenopsis. In this study, we cloned 12 candidate reference genes including 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), cytoskeletal structural protein actin (ACT1, ACT2, ACT3, ACT4, ACT5), ubiquitin protein (UBQ1 and UBQ2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the cytoskeletal structural proteins α-tubulin (TUA) and β-tubulin (TUB) in Phalaenopsis and evaluated their expression reliability. The expression of these candidate reference genes was analyzed using geNorm and normFinder software packages; the results showed that ACT2 and ACT4 were the highest stability reference genes for all experiment sets based on normFinder, followed by ACT1 or ACT3, while ACT3 and ACT4 were the highest stability reference genes for most experiment sets based on geNorm, then TUB or others. Taken together, Actin genes were the higher stability reference genes for all tissues at total developmental stages, and similar results came from analysis by normFinder. According to geNorm analysis, ACT3 and ACT4 were the most stable reference genes for all tissues tested and tissues at reproductive stages; TUB and ACT5 or ACT4 were the most stable reference genes for vegetative tissues or roots. The most stable reference genes for all vegetative tissues and only leaves were ACT4 and ACT5, ACT2 and ACT3, respectively; ACT1 and ACT3 were the most stable genes and sufficient for reliable normalization of flower tissues. While EF1α, UBQ1, UBQ2, and GAPDH were found to be unsuitable as a reference gene in our analysis for flower tissues, total tissues, and reproductive stages; UBQ2 and 18S were identified as the least stable reference genes for vegetative tissues at different stages, different tissues at vegetative stages; TUA and 18S were the

  2. Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction.

    PubMed

    Pinches, Mark D G; Helps, Christopher R; Gruffydd-Jones, Tim J; Egan, Kathy; Jarrett, Oswald; Tasker, Séverine

    2007-02-01

    In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.

  3. Quantitative Real-Time PCR Analysis of Gene Transcripts of Mosquito Follicles.

    PubMed

    Telang, Aparna

    2016-01-01

    Real-time (quantitative) PCR, or QPCR, has become an indispensible tool for characterizing gene expression. Depending on the experimental design, researchers can use either the relative or absolute (standard curve) method to quantify transcript abundance. Characterizing the expression of genes in mosquito ovaries will require use of the standard curve method of quantification. Here, I describe reagents and equipment necessary to run standard curve QPCR. I also provide details on the construction of the standard linear curve and calculations required to determine transcript abundance.

  4. Sensitive on-chip quantitative real-time PCR performed on an adaptable and robust platform.

    PubMed

    Lund-Olesen, Torsten; Dufva, Martin; Dahl, John Arne; Collas, Philippe; Hansen, Mikkel Fougt

    2008-12-01

    A robust, flexible and efficient system for performing high sensitivity quantitative on-chip real-time PCR for research purposes is presented. The chips used consist of microchannels etched in silicon. The surface in the channels is a thermally grown silicon dioxide and the channel is sealed by a glass lid. The chips contain four PCR chambers but this number can be increased for further multiplexing. Contrary to PCR chips with oil covered open chambers, these channel-like chambers are easily integrated in lab-on-a-chip devices. The temperature is controlled by a Peltier element and the fluorochrome detector system is a commercially available fluorescence stereo microscope equipped with a CCD camera. The setup shows an excellent signal-to-noise ratio of about 400 compared to that of about 150 obtained in a commercial real time PCR machine. A detection limit of a few copies of target molecules is found, which is 100 to 100,000-fold better than other on-chip real-time PCR systems presented in the literature. This demonstrates that the PCR system can be used for critical applications. We also demonstrate that high quality melting curves can be obtained. Such curves are important in lab-on-a-chip systems for identification of amplified product. The usability of the system is validated by performing quantitative on-chip measurements of the amount of specific gene sequences co-immunoprecipitated with various posttranslationally modified histone proteins. Similar results are obtained from on-chip experiments and experiments carried out in a commercial system on larger sample volumes.

  5. Real-time quantitative fluorescence measurement of microscale cell culture analog systems

    NASA Astrophysics Data System (ADS)

    Oh, Taek-il; Kim, Donghyun; Tatosian, Daniel; Sung, Jong Hwan; Shuler, Michael

    2007-02-01

    A microscale cell culture analog (μCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A μCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A μCCA is expected to provide a tool for high-throughput drug discovery. Previously, a portable fluorescence detection system was investigated for a single μCCA device in real-time. In this study, we present a fluorescence-based imaging system that provides quantitative real-time data of the metabolic interactions in μCCAs with an emphasis on measuring multiple μCCA samples simultaneously for high-throughput screening. The detection system is based on discrete optics components, with a high-power LED and a charge-coupled device (CCD) camera as a light source and a detector, for monitoring cellular status on the chambers of each μCCA sample. Multiple samples are characterized mechanically on a motorized linear stage, which is fully-automated. Each μCCA sample has four chambers, where cell lines MES-SA/DX- 5, and MES-SA (tumor cells of human uterus) have been cultured. All cell-lines have been transfected to express the fusion protein H2B-GFP, which is a human histone protein fused at the amino terminus to EGFP. As a model cytotoxic drug, 10 μM doxorubicin (DOX) was used. Real-time quantitative data of the intensity loss of enhanced green fluorescent protein (EGFP) during cell death of target cells have been collected over several minutes to 40 hours. Design issues and improvements are also discussed.

  6. Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

    PubMed Central

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

  7. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV).

    PubMed

    Purcell, Maureen K; Thompson, Rachel L; Garver, Kyle A; Hawley, Laura M; Batts, William N; Sprague, Laura; Sampson, Corie; Winton, James R

    2013-10-11

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  8. Real-Time Fluorogenic Reverse Transcription-PCR Assays for Detection of Bacteriophage MS2

    PubMed Central

    O'Connell, Kevin P.; Bucher, Jennifer R.; Anderson, Patricia E.; Cao, Cheng J.; Khan, Akbar S.; Gostomski, Mark V.; Valdes, James J.

    2006-01-01

    Bacteriophage MS2 is used in place of pathogenic viruses in a wide variety of studies that range from testing of compounds for disinfecting surfaces to studying environmental transport and fate of pathogenic viruses in groundwater. MS2 is also used as a pathogen simulant in the research, development, and testing (including open air tests) of methods, systems, and devices for the detection of pathogens in both the battlefield and homeland defense settings. PCR is often used as either an integral part of such detection systems or as a reference method to assess the sensitivity and specificity of microbial detection. To facilitate the detection of MS2 by PCR, we describe here a set of real-time fluorogenic reverse transcription-PCR assays. The sensitivity of the assays (performed with primer pairs and corresponding dye-labeled probes) ranged from 0.4 to 40 fg of MS2 genomic RNA (200 to 20,000 genome equivalents). We also demonstrate the usefulness of the primer pairs in assays without dye-labeled probe that included the DNA-binding dye SYBR green. None of the assays gave false-positive results when tested against 400 pg of several non-MS2 nucleic acid targets. PMID:16391081

  9. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

    USGS Publications Warehouse

    Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

    2013-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  10. Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus real-time PCR.

    PubMed

    Jenkins, Andrew; Allum, Anne-Gry; Strand, Linda; Aakre, Randi Kersten

    2013-02-01

    A consensus multiplex real-time PCR test (PT13-RT) for the oncogenic human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 is described. The test targets the L1 gene. Analytical sensitivity is between 4 and 400 GU (genomic units) in the presence of 500 ng of human DNA, corresponding to 75,000 human cells. HPV types are grouped into multiplex groups of 3 or 4 resulting in the use of 4 wells per sample and permitting up to 24 samples per run (including controls) in a standard 96-well real-time PCR instrument. False negative results are avoided by (a) measuring sample DNA concentration to control that sufficient cellular material is present and (b) including HPV type 6 as a homologous internal control in order to detect PCR inhibition or competition from other (non-oncogenic) HPV types. Analysis time from refrigerator to report is 8 h, including 2.5 h hands-on time. Relative to the HC2 test, the sensitivity and specificity were respectively 98% and 83%, the lower specificity being attributable to the higher analytical sensitivity of PT13-RT. To assess type determination comparison was made with a reversed line-blot test. Type concordance was high (κ=0.79) with discrepancies occurring mostly in multiple-positive samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Reference gene selection for real-time quantitative PCR analysis on ovarian cryopreservation by vitrification in mice.

    PubMed

    Yuanyuan, Shan; Qin, Su; Rongrong, Xu; Yujing, Gao; Chengbin, Pei; Jianjun, Ma; Yanzhou, Yang; Xiuying, Pei

    2015-08-01

    To ensure the correct interpretation of the results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) from ovarian tissue cryopreserved by vitrification, it is critical to normalize expression levels to a reference gene with stable messenger RNA (mRNA) expression in the vitrified/warmed ovarian tissue. The aim of this work was to identify suitable reference genes for qRT-PCR analysis during ovarian cryopreservation by vitrification. GeNorm, NormFinder, comparative Delta-CT, and BestKeeper were used to analyze the expression and stability of the 14 reference genes GAPDH, ABL1, ACTB, CDKN1A, GPER, GUSB, HPRT1, HSP90AB1, IPO8, PPIA, RPL4, RPL30, TBP, and UPAR. Our results indicated that ACTB and RPL4 were relatively stable reference genes in vitrified/warmed ovaries.

  12. Development of two quantitative real-time PCR diagnostic kits for HPV isolates from Korea.

    PubMed

    Jeeva, Subbiah; Kim, Nam-Il; Jang, In-Kwon; Choi, Tae-Jin

    2012-10-01

    Viral pathogens, alongside other pathogens, have major effects on crustacean aquaculture. Hepatopancreatic parvovirus (HPV) is an emerging virus in the shrimp industry and has been detected in shrimp farms worldwide. The HPV genome has greater diversity than other shrimp viruses owing to its wide host range and geographical distribution. Therefore, developing diagnostic tools is essential to detect even small copy numbers from the target region of native HPV isolates. We have developed two easy to use quantitative real-time PCR kits, called Green Star and Dual Star, which contain all of the necessary components for real-time PCR, including HPV primers, using the primers obtained from the sequences of HPV isolates from Korea, and analyzed their specificity, efficiency, and reproducibility. These two kits could detect from 1 to 1 × 10(9) copies of cloned HPV DNA. The minimum detection limits obtained from HPV-infected shrimp were 7.74 × 10(1) and 9.06 × 10(1) copies in the Green Star and Dual Star assay kits, respectively. These kits can be used for rapid, sensitive, and efficient screening for HPV isolates from Korea before the introduction of postlarval stages into culture ponds, thereby decreasing the incidence of early development of the disease.

  13. Improved radar data processing algorithms for quantitative rainfall estimation in real time.

    PubMed

    Krämer, S; Verworn, H R

    2009-01-01

    This paper describes a new methodology to process C-band radar data for direct use as rainfall input to hydrologic and hydrodynamic models and in real time control of urban drainage systems. In contrast to the adjustment of radar data with the help of rain gauges, the new approach accounts for the microphysical properties of current rainfall. In a first step radar data are corrected for attenuation. This phenomenon has been identified as the main cause for the general underestimation of radar rainfall. Systematic variation of the attenuation coefficients within predefined bounds allows robust reflectivity profiling. Secondly, event specific R-Z relations are applied to the corrected radar reflectivity data in order to generate quantitative reliable radar rainfall estimates. The results of the methodology are validated by a network of 37 rain gauges located in the Emscher and Lippe river basins. Finally, the relevance of the correction methodology for radar rainfall forecasts is demonstrated. It has become clearly obvious, that the new methodology significantly improves the radar rainfall estimation and rainfall forecasts. The algorithms are applicable in real time.

  14. Enabling reproducible real-time quantitative PCR research: the RDML package.

    PubMed

    Rödiger, Stefan; Burdukiewicz, Michal; Spiess, Andrej-Nikolai; Blagodatskikh, Konstantin

    2017-08-26

    Reproducibility, a cornerstone of research, requires defined data formats, which include the set-up and output of experiments. The Real-time PCR Data Markup Language (RDML) is a recommended standard of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Despite the popularity of the RDML format for analysis of qPCR data, handling of RDML files is not yet widely supported in all PCR curve analysis softwares. This study describes the open source RDML package for the statistical computing language RDML is compatible with RDML versions ≤ 1.2 and provides functionality to (i) import RDML data; (ii) extract sample information (e.g., targets, concentration); (iii) transform data to various formats of the environment; (iv) generate human readable run summaries; and (v) to create RDML files from user data. In addition, RDML offers a graphical user interface to read, edit and create RDML files. https://cran.r-project.org/package=RDML . rdmlEdit server http://shtest.evrogen.net/rdmlEdit/ . Documentation: http://kablag.github.io/RDML/ . k.blag@yandex.ru. Supplementary data are available at Bioinformatics online.

  15. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.

    PubMed

    Bae, Hi-Gung; Nitsche, Andreas; Teichmann, Anette; Biel, Stefan S; Niedrig, Matthias

    2003-06-30

    Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).

  16. Validation of absolute quantitative real-time PCR for the diagnosis of Streptococcus agalactiae in fish.

    PubMed

    Sebastião, Fernanda de A; Lemos, Eliana G M; Pilarski, Fabiana

    2015-12-01

    Streptococcus agalactiae (GBS) are Gram-positive cocci responsible for substantial losses in tilapia fish farms in Brazil and worldwide. It causes septicemia, meningoencephalitis and mortality of whole shoals that can occur within 72 h. Thus, diagnostic methods are needed that are rapid, specific and sensitive. In this study, a pair of specific primers for GBS was generated based on the cfb gene sequence and initially evaluated by conventional PCR. The protocols for absolute quantitative real-time PCR (qPCR) were then adapted to validate the technique for the identification and quantification of GBS isolated by real-time detection of amplicons using fluorescence measurements. Finally, an infectivity test was conducted in tilapia infected with GBS strains. Total DNA from the host brain was subjected to the same technique, and the strains were re-isolated to validate Koch's postulates. The assay showed 100% specificity for the other bacterial species evaluated and a sensitivity of 367 gene copies per 20 mg of brain tissue within 4 h, making this test a valuable tool for health monitoring programs. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

    PubMed Central

    Abt, Melissa A.; Grek, Christina L.; Ghatnekar, Gautam S.; Yeh, Elizabeth S.

    2016-01-01

    Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3–6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue. PMID:26862835

  18. Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR.

    PubMed

    Abt, Melissa A; Grek, Christina L; Ghatnekar, Gautam S; Yeh, Elizabeth S

    2016-01-29

    Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death. Common sites of metastatic spread include lung, lymph node, brain, and bone. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

  19. Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

    PubMed

    Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

    2008-12-01

    One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

  20. Immuno-laser capture microdissection of rat brain neurons for real time quantitative PCR.

    PubMed

    Baskin, Denis G; Bastian, L Scot

    2010-01-01

    Laser capture microdissection (LCM) is a technical approach for obtaining microscopic samples as small as individual cells from tissues for molecular analysis. While the principles and details of the operation of LCM instruments, the technical requirements for obtaining identified cells for LCM "picking", all share the common feature of using a laser in combination with a microscope to microdissect and remove cells from tissue slices (or cultured cells) mounted on a glass slide. The use of LCM is becoming widespread in pathology laboratories and is increasingly being used for gene expression studies in cell biology. The approach is particularly powerful when used in conjunction with immunostaining techniques to obtain enriched RNA samples from cells that have been collected by picking and gathering phenotypically similar cells from anatomically complex organs such as the brain. In the present chapter, we describe an approach for combining immunocytochemistry with LCM to obtain RNA for real time quantitative PCR.

  1. Evaluation of postmortem bacterial migration using culturing and real-time quantitative PCR.

    PubMed

    Tuomisto, Sari; Karhunen, Pekka J; Vuento, Risto; Aittoniemi, Janne; Pessi, Tanja

    2013-07-01

    Postmortem bacteriology can be a valuable tool for evaluating deaths due to bacterial infection or for researching the involvement of bacteria in various diseases. In this study, time-dependent postmortem bacterial migration into liver, mesenteric lymph node, pericardial fluid, portal, and peripheral vein was analyzed in 33 autopsy cases by bacterial culturing and real-time quantitative polymerase chain reaction (RT-qPCR). None suffered or died from bacterial infection. According to culturing, pericardial fluid and liver were the most sterile samples up to 5 days postmortem. In these samples, multigrowth and staphylococci were not or rarely detected. RT-qPCR was more sensitive and showed higher bacterial positivity in all samples. Relative amounts of intestinal bacterial DNA (bifidobacteria, bacteroides, enterobacter, clostridia) increased with time. Sterility of blood samples was low during the studied time periods (1-7 days). The best postmortem microbiological sampling sites were pericardial fluid and liver up to 5 days after death.

  2. Real time quantitative imaging for semiconductor crystal growth, control and characterization

    NASA Technical Reports Server (NTRS)

    Wargo, Michael J.

    1991-01-01

    A quantitative real time image processing system has been developed which can be software-reconfigured for semiconductor processing and characterization tasks. In thermal imager mode, 2D temperature distributions of semiconductor melt surfaces (900-1600 C) can be obtained with temperature and spatial resolutions better than 0.5 C and 0.5 mm, respectively, as demonstrated by analysis of melt surface thermal distributions. Temporal and spatial image processing techniques and multitasking computational capabilities convert such thermal imaging into a multimode sensor for crystal growth control. A second configuration of the image processing engine in conjunction with bright and dark field transmission optics is used to nonintrusively determine the microdistribution of free charge carriers and submicron sized crystalline defects in semiconductors. The IR absorption characteristics of wafers are determined with 10-micron spatial resolution and, after calibration, are converted into charge carrier density.

  3. Real time quantitative imaging for semiconductor crystal growth, control and characterization

    NASA Technical Reports Server (NTRS)

    Wargo, Michael J.

    1991-01-01

    A quantitative real time image processing system has been developed which can be software-reconfigured for semiconductor processing and characterization tasks. In thermal imager mode, 2D temperature distributions of semiconductor melt surfaces (900-1600 C) can be obtained with temperature and spatial resolutions better than 0.5 C and 0.5 mm, respectively, as demonstrated by analysis of melt surface thermal distributions. Temporal and spatial image processing techniques and multitasking computational capabilities convert such thermal imaging into a multimode sensor for crystal growth control. A second configuration of the image processing engine in conjunction with bright and dark field transmission optics is used to nonintrusively determine the microdistribution of free charge carriers and submicron sized crystalline defects in semiconductors. The IR absorption characteristics of wafers are determined with 10-micron spatial resolution and, after calibration, are converted into charge carrier density.

  4. Development of a real-time quantitative PCR assay to enumerate Yersinia pestis in fleas.

    PubMed

    Gabitzsch, Elizabeth S; Vera-Tudela, Rommelle; Eisen, Rebecca J; Bearden, Scott W; Gage, Kenneth L; Zeidner, Nordin S

    2008-07-01

    A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.

  5. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    NASA Astrophysics Data System (ADS)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  6. New Developments in Quantitative Real-time Polymerase Chain Reaction Technology.

    PubMed

    Gadkar, Vija yJ; Filion, Martin

    2014-01-01

    Real time-quantitative PCR (RT-qPCR) technology has revolutionized the detection landscape in every area of molecular biology. The fundamental basis of this technology has remained unchanged since its inception, however various modifications have enhanced the overall performance of this highly versatile technology. These improvements have ranged from changes in the individual components of the enzymatic reaction cocktail (polymerizing enzymes, reaction buffers, probes, etc.) to the detection system itself (instrumentation, software, etc.). The RT-qPCR technology currently available to researchers is more sensitive, faster and affordable than when this technology was first introduced. In this article, we summarize the developments of the last few years in RT-qPCR technology and nucleic acid amplification.

  7. Reverse transcription real-time PCR for detection of porcine interferon α and β genes.

    PubMed

    Razzuoli, E; Villa, R; Sossi, E; Amadori, M

    2011-10-01

    A few studies provided convincing evidence of constitutive expression of type I interferons (IFNs) in humans and mice, and of the steady-state role of these cytokines under health conditions. These results were later confirmed in pigs, too. In line with this tenet, low levels of IFN-α/β can be detected in swine tissues in the absence of any specific inducer. These studies are compounded by the utmost complexity of type I IFNs (including among others 17 IFN-α genes in pigs), which demands proper research tools. This prompted us to analyse the available protocols and to develop a relevant, robust, reverse transcription (RT) real-time polymerase chain reaction (PCR) detection system for the amplification of porcine IFN-α/β genes. The adopted test procedure is user-friendly and provides the complete panel of gene expression of one subject in a microtitre plate. Also, a proper use of PCR fluorochromes (SYBR(®) versus EvaGreen(®) supermix) enables users to adopt proper test protocols in case of low-expression porcine IFN-α genes. This is accounted for by the much higher sensitivity of the test protocol with EvaGreen(®) supermix. Interestingly, IFN-β showed the highest frequency of constitutive expression, in agreement with its definition of 'immediate early' gene in both humans and mice. Results indicate that the outlined procedure can detect both constitutively expressed and virus-induced IFN-α/β genes, as well as the impact of environmental, non-infectious stressors on the previous profile of constitutive expression.

  8. Real-Time Reverse Transcription-PCR Assay Panel for Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Lu, Xiaoyan; Whitaker, Brett; Sakthivel, Senthil Kumar K.; Kamili, Shifaq; Rose, Laura E.; Lowe, Luis; Mohareb, Emad; Elassal, Emad M.; Al-sanouri, Tarek; Haddadin, Aktham

    2014-01-01

    A new human coronavirus (CoV), subsequently named Middle East respiratory syndrome (MERS)-CoV, was first reported in Saudi Arabia in September 2012. In response, we developed two real-time reverse transcription-PCR (rRT-PCR) assays targeting the MERS-CoV nucleocapsid (N) gene and evaluated these assays as a panel with a previously published assay targeting the region upstream of the MERS-CoV envelope gene (upE) for the detection and confirmation of MERS-CoV infection. All assays detected ≤10 copies/reaction of quantified RNA transcripts, with a linear dynamic range of 8 log units and 1.3 × 10−3 50% tissue culture infective doses (TCID50)/ml of cultured MERS-CoV per reaction. All assays performed comparably with respiratory, serum, and stool specimens spiked with cultured virus. No false-positive amplifications were obtained with other human coronaviruses or common respiratory viral pathogens or with 336 diverse clinical specimens from non-MERS-CoV cases; specimens from two confirmed MERS-CoV cases were positive with all assay signatures. In June 2012, the U.S. Food and Drug Administration authorized emergency use of the rRT-PCR assay panel as an in vitro diagnostic test for MERS-CoV. A kit consisting of the three assay signatures and a positive control was assembled and distributed to public health laboratories in the United States and internationally to support MERS-CoV surveillance and public health responses. PMID:24153118

  9. Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children

    PubMed Central

    Song, Yuli; Liu, Chengxu; Finegold, Sydney M.

    2004-01-01

    Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 × 108 CFU/g in autistic children and 4.8 × 108 CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract. PMID:15528506

  10. Investigation of telomere lengths measurement by quantitative real-time PCR to predict age.

    PubMed

    Hewakapuge, Sudinna; van Oorschot, Roland A H; Lewandowski, Paul; Baindur-Hudson, Swati

    2008-09-01

    Currently DNA profiling methods only compare a suspect's DNA with DNA left at the crime scene. When there is no suspect, it would be useful for the police to be able to predict what the person of interest looks like by analysing the DNA left behind in a crime scene. Determination of the age of the suspect is an important factor in creating an identikit. Human somatic cells gradually lose telomeric repeats with age. This study investigated if one could use a correlation between telomere length and age, to predict the age of an individual from their DNA. Telomere length, in buccal cells, of 167 individuals aged between 1 and 96 years old was measured using real-time quantitative PCR. Telomere length decreased with age (r=-0.185, P<0.05) and the age of an individual could be roughly determined by the following formula: (age=relative telomere length -1.5/-0.005). The regression (R(2)) value between telomere length and age was approximately 0.04, which is too low to be use for forensics. The causes for the presence of large variation in telomere lengths in the population were further investigated. The age prediction accuracies were low even after dividing samples into non-related Caucasians, males and females (5%, 9% and 1%, respectively). Mean telomere lengths of eight age groups representing each decade of life showed non-linear decrease in telomere length with age. There were variations in telomere lengths even among similarly aged individuals aged 26 years old (n=10) and age 54 years old (n=9). Therefore, telomere length measurement by real-time quantitative PCR cannot be used to predict age of a person, due to the presence of large inter-individual variations in telomere lengths.

  11. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143

  12. Evaluation of residual antibacterial potency in antibiotic production wastewater using a real-time quantitative method.

    PubMed

    Zhang, Hong; Zhang, Yu; Yang, Min; Liu, Miaomiao

    2015-11-01

    While antibiotic pollution has attracted considerable attention due to its potential in promoting the dissemination of antibiotic resistance genes in the environment, the antibiotic activity of their related substances has been neglected, which may underestimate the environmental impacts of antibiotic wastewater discharge. In this study, a real-time quantitative approach was established to evaluate the residual antibacterial potency of antibiotics and related substances in antibiotic production wastewater (APW) by comparing the growth of a standard bacterial strain (Staphylococcus aureus) in tested water samples with a standard reference substance (e.g. oxytetracycline). Antibiotic equivalent quantity (EQ) was used to express antibacterial potency, which made it possible to assess the contribution of each compound to the antibiotic activity in APW. The real-time quantitative method showed better repeatability (Relative Standard Deviation, RSD 1.08%) compared with the conventional fixed growth time method (RSD 5.62-11.29%). And its quantification limits ranged from 0.20 to 24.00 μg L(-1), depending on the antibiotic. We applied the developed method to analyze the residual potency of water samples from four APW treatment systems, and confirmed a significant contribution from antibiotic transformation products to potent antibacterial activity. Specifically, neospiramycin, a major transformation product of spiramycin, was found to contribute 13.15-22.89% of residual potency in spiramycin production wastewater. In addition, some unknown related substances with antimicrobial activity were indicated in the effluent. This developed approach will be effective for the management of antibacterial potency discharge from antibiotic wastewater and other waste streams.

  13. Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR.

    PubMed

    Benassi, Julia Cristina; Benvenga, Graziella U; Ferreira, Helena Lage; Pereira, Vanessa F; Keid, Lara B; Soares, Rodrigo; Oliveira, Tricia Maria Ferreira de Sousa

    2017-06-01

    Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla

    PubMed Central

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates – five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) – using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔCt, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions. PMID:27446172

  15. Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR.

    PubMed

    Smith, Jeffery L; Rangaraj, Kavitha; Simpson, Robert; Maclean, Donald J; Nathanson, Les K; Stuart, Katherine A; Scott, Shaun P; Ramm, Grant A; de Jersey, John

    2004-04-01

    ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to beta-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.

  16. Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus

    USDA-ARS?s Scientific Manuscript database

    A subtype specific H7 real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay developed by the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 in North and South American wild aquatic birds and poultry was validated as a collaborative effort by the SEPRL and Na...

  17. Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay

    PubMed Central

    Savage, Harry M.

    2017-01-01

    We assayed Zika virus–infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days. PMID:28075325

  18. Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay.

    PubMed

    Burkhalter, Kristen L; Savage, Harry M

    2017-04-01

    We assayed Zika virus-infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days.

  19. Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

    PubMed

    Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

    2011-07-01

    Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

  20. Simultaneous detection of influenza viruses A and B using real-time quantitative PCR.

    PubMed

    van Elden, L J; Nijhuis, M; Schipper, P; Schuurman, R; van Loon, A M

    2001-01-01

    Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

  1. Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

    PubMed Central

    Loftie-Eaton, Wesley; Tucker, Allison; Norton, Ann

    2014-01-01

    The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses. PMID:24973062

  2. Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments.

    PubMed

    Klenke, Stefanie; Renckhoff, Kristina; Engler, Andrea; Peters, Jürgen; Frey, Ulrich H

    2016-12-01

    Real-time PCR is an indispensable technique for mRNA expression analysis but conclusions depend on appropriate reference gene selection. However, while reference gene selection has been a topic of publications, this issue is often disregarded when measuring target mRNA expression. Therefore, we (1) evaluated the frequency of appropriate reference gene selection, (2) suggest an easy-to-use tool for least variability reference gene selection, (3) demonstrate application of this tool, and (4) show effects on target gene expression profiles. All 2015 published articles in Naunyn-Schmiedeberg's Archives of Pharmacology were screened for the use of quantitative real-time PCR analysis and selection of reference genes. Target gene expression (Vegfa, Grk2, Sirt4, and Timp3) in H9c2 cells was analyzed following various interventions (hypoxia, hyperglycemia, and/or isoflurane exposure with and without subsequent hypoxia) in relation to putative reference genes (Actb, Gapdh, B2m, Sdha, and Rplp1) using the least variability method vs. an arbitrarily selected but established reference gene. In the vast majority (18 of 21) of papers, no information was provided regarding selection of an appropriate reference gene. In only 1 of 21 papers, a method of appropriate reference gene selection was described and in 2 papers reference gene selection remains unclear. The method of reference gene selection had major impact on interpretation of target gene expression. With hypoxia, for instance, the least variability gene was Rplp1 and target gene expression (Vefga) heavily showed a 2-fold up-regulation (p = 0.022) but no change (p = 0.3) when arbitrarily using Gapdh. Frequency of appropriate reference gene selection in this journal is low, and we propose our strategy for reference gene selection as an easy tool for proper target gene expression.

  3. Development of SYBR Green real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus.

    PubMed

    Park, Sang-Ik; Park, Da-Hae; Saif, Linda J; Jeong, Young-Ju; Shin, Dong-Jun; Chun, Young-Hyun; Park, Su-Jin; Kim, Hyun-Jeong; Hosmillo, Myra; Kwon, Hyung-Jun; Kang, Mun-Il; Cho, Kyoung-Oh

    2009-07-01

    Unclassified bovine enteric calicivirus (BECV) is a newly recognized bovine enteric calicivirus that differs from bovine norovirus, and which causes diarrhea in the small intestines of calves. To date, methods such as real-time reverse transcription-polymerase chain reaction (RT-PCR) have not been developed for the rapid detection, quantitation and diagnosis of BECV. Presently, a BECV-specific SYBR Green real-time RT-PCR assay was evaluated and optimized. Diarrheic specimens (n=118) collected from 2004 to 2005 were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR and nested PCR, 9 (7.6%) and 59 (50%) samples tested positive, respectively, whereas the SYBR Green assay detected BECV in 91 (77.1%) samples. Using BECV RNA standards generated by in vitro transcription, the SYBR Green real-time RT-PCR assay sensitively detected BECV RNA to 1.1 x 10(0)copies/microl (correlation coefficiency=0.98). The detection limits of the RT-PCR and nested PCR were 1.1 x 10(5) and 1.1 x 10(2)copies/microl, respectively. These results indicate that the SYBR Green real-time RT-PCR assay is more sensitive than conventional RT-PCR and nested PCR assays, and has potential as a reliable, reproducible, specific, sensitive and rapid tool for the detection, quantitation and diagnosis of unclassified BECV.

  4. Comparison of Analytic Methods for Quantitative Real-Time Polymerase Chain Reaction Data

    PubMed Central

    Chen, Ping

    2015-01-01

    Abstract Polymerase chain reaction (PCR) is a laboratory procedure to amplify and simultaneously quantify targeted DNA molecules, and then detect the product of the reaction at the end of all the amplification cycles. A more modern technique, real-time PCR, also known as quantitative PCR (qPCR), detects the product after each cycle of the progressing reaction by applying a specific fluorescence technique. The quantitative methods currently used to analyze qPCR data result in varying levels of estimation quality. This study compares the accuracy and precision of the estimation achieved by eight different models when applied to the same qPCR dataset. Also, the study evaluates a newly introduced data preprocessing approach, the taking-the-difference approach, and compares it to the currently used approach of subtracting the background fluorescence. The taking-the-difference method subtracts the fluorescence in the former cycle from that in the latter cycle to avoid estimating the background fluorescence. The results obtained from the eight models show that taking-the-difference is a better way to preprocess qPCR data compared to the original approach because of a reduction in the background estimation error. The results also show that weighted models are better than non-weighted models, and that the precision of the estimation achieved by the mixed models is slightly better than that achieved by the linear regression models. PMID:26204477

  5. Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction.

    PubMed

    von Kanel, Thomas; Gerber, Dominik; Wittwer, Carl T; Hermann, Mark; Gallati, Sabina

    2011-12-15

    Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Comparison of analytic methods for quantitative real-time polymerase chain reaction data.

    PubMed

    Chen, Ping; Huang, Xuelin

    2015-11-01

    Polymerase chain reaction (PCR) is a laboratory procedure to amplify and simultaneously quantify targeted DNA molecules, and then detect the product of the reaction at the end of all the amplification cycles. A more modern technique, real-time PCR, also known as quantitative PCR (qPCR), detects the product after each cycle of the progressing reaction by applying a specific fluorescence technique. The quantitative methods currently used to analyze qPCR data result in varying levels of estimation quality. This study compares the accuracy and precision of the estimation achieved by eight different models when applied to the same qPCR dataset. Also, the study evaluates a newly introduced data preprocessing approach, the taking-the-difference approach, and compares it to the currently used approach of subtracting the background fluorescence. The taking-the-difference method subtracts the fluorescence in the former cycle from that in the latter cycle to avoid estimating the background fluorescence. The results obtained from the eight models show that taking-the-difference is a better way to preprocess qPCR data compared to the original approach because of a reduction in the background estimation error. The results also show that weighted models are better than non-weighted models, and that the precision of the estimation achieved by the mixed models is slightly better than that achieved by the linear regression models.

  7. On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets

    SciTech Connect

    Beer, N R; Wheeler, E; Lee-Houghton, L; Watkins, N; Nasarabadi, S; Hebert, N; Leung, P; Arnold, D; Bailey, C; Colston, B

    2007-12-19

    The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment, and will be useful in viral discovery and gene-profiling applications.

  8. Real-Time, In Vivo Monitoring, and Quantitative Assessment of Intra-Arterial Vasospasm Therapy.

    PubMed

    Gölitz, Philipp; Kaschka, Iris; Lang, Stefan; Roessler, Karl; Knossalla, Frauke; Doerfler, Arnd

    2016-08-01

    Our study aimed to evaluate whether the effect of an intra-arterial vasospasm therapy can be assessed quantitatively by in vivo blood flow analysis using the postprocessing algorithm parametric color coding (PCC). We evaluated 17 patients presenting with acute clinical deterioration due to vasospasm following subarachnoidal hemorrhage treated with intra-arterial nimodipine application. Pre- and post-interventional DSA series were post-processed by PCC. The relative time to maximum opacification (rTmax) was calculated in 14 arterially and venously located points of interest. From that data, the pre- and post-interventional cerebral circulation time (CirT) was calculated. Additionally, the arterial vessel diameters were measured. Pre- and post-interventional values were compared and tested for significance, respectively. Flow analysis revealed in all arterial vessel segments a non-statistically significant prolongation of rTmax after treatment. The mean CirT was 5.62 s (±1.19 s) pre-interventionally and 5.16 s (±0.81 s) post-interventionally, and the difference turned out as statistically significant (p = 0.039). A significantly increased diameter was measurable in all arterial segments post-interventionally. PCC is a fast applicable imaging technique that allows via real-time and in vivo blood flow analysis a quantitative assessment of the effect of intra-arterial vasospasm therapy. Our results seem to validate in vivo that an intra-arterial nimodipine application induces not only vasodilatation of the larger vessels, but also improves the microcirculatory flow, leading to a shortened cerebral CirT that reaches normal range post-interventionally. Procedural monitoring via PCC offers the option to compare quantitatively different therapy regimes, which allows optimization of existing approaches and implementation of individualized treatment strategies.

  9. Assessment of the inhibitory effect of ribavirin on the rainbow trout rhabdovirus VHSV by real-time reverse-transcription PCR.

    PubMed

    Marroquí, Laura; Estepa, Amparo; Perez, Luis

    2007-05-16

    Viral hemorrhagic septicemia virus (VHSV) is one of the most ubiquitous viruses in salmonid aquaculture in Europe. This infectious disease results in significant losses in the farming industry and therefore effective therapeutic agents are needed to control outbreaks caused by this pathogen. Thus, accurate methods to test new antiviral compounds need to be developed. Our goal was to establish a model system for testing novel antivirals with potential applications to aquaculture. In a previous study, a TaqMan real-time RT-PCR assay was designed to detect and quantitate VHSV in rainbow trout tissues [Chico, V., Gomez, N., Estepa, A., Perez, L., 2006. Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J. Virol. Methods 132, 154-159]. In this report, we applied the real-time RT-PCR assay to the evaluation of the inhibitory effect of ribavirin, a well-known broad spectrum antiviral drug, in a cell culture system. When added from the beginning of the infection, ribavirin caused a dose-dependent reduction of VHSV RNA accumulation. Real-time RT-PCR measurements showed 99.8% inhibition at 25 microg/ml ribavirin, with an IC50 of 0.43 microg/ml. Ribavirin maintained its inhibitory activity against VHSV when added at 6 h post-infection. Quantitation of N protein messenger RNA and plus-stranded RNA showed a substantial decrease of viral transcription in ribavirin-treated cells. Partial reversion of the effect of ribavirin by addition of GTP was observed, confirming that ribavirin targets the synthesis of guanidine nucleotides in the cells. This is the first report of a real-time PCR-based assay for addressing the efficacy and mechanism of action of an antiviral agent for rainbow trout.

  10. EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER

    EPA Science Inventory

    Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.

    Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR)...

  11. EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER

    EPA Science Inventory

    Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.

    Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR)...

  12. Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.

    PubMed

    Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

    2016-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

  13. Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum

    PubMed Central

    Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

    2016-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels. PMID:27942007

  14. Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR.

    PubMed

    Hirai, Keiji; Yoshizawa, Hiromichi; Imai, Toshimi; Igarashi, Yusuke; Hirahara, Ichiro; Ookawara, Susumu; Ishibashi, Kenichi; Morishita, Yoshiyuki

    2017-06-27

    MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

  15. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  16. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation.

    PubMed

    Wei, Yongcheng; Liu, Qinghua; Dong, Hongyu; Zhou, Zhichun; Hao, Yanping; Chen, Xuelian; Xu, Liuyi

    2016-01-01

    Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.

  17. Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

    PubMed

    Neumann, A P; Dunham, S M; Rehberger, T G; Siragusa, G R

    2010-08-01

    Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.

  18. Utility of Real-Time Quantitative Polymerase Chain Reaction in Detecting Mycobacterium tuberculosis

    PubMed Central

    Zhang, Mingxin; Zhang, Hui

    2017-01-01

    This study aimed to assess the value of real-time quantitative polymerase chain reaction (RT-qPCR) for the detection of Mycobacterium tuberculosis (MTB). Samples from 192 patients with suspected MTB were examined by RT-qPCR and an improved Löwenstein–Jensen (L-J) culture method. To evaluate the diagnostic usefulness of RT-qPCR in detecting MTB, a receiver operating characteristic (ROC) curve for RT-qPCR was generated, and the area under the curve (AUC) as well as a cutoff value was calculated. Using the L-J culture method as the gold standard, accuracy of the RT-qPCR method for detecting MTB was 92.7%, with sensitivity and specificity of 62.5% and 97.02%, respectively. In comparison with the improved L-J culture method, the AUC of RT-qPCR ROC curve was 0.957, which was statistically significant (p < 0.001). The Youden Index reached the maximum value (0.88) for gene copy number of 794.5 IU/mL, which was used as the cutoff value. RT-qPCR detection of MTB yielded results consistent with those of the improved L-J culture method, with high accuracy. RT-qPCR may be used as an auxiliary method for etiological diagnosis of tuberculosis. PMID:28168192

  19. Real-time PCR for quantitative detection of bovine tissues in food and feed.

    PubMed

    Martín, Irene; García, Teresa; Fajardo, Violeta; Rojas, María; Hernández, Pablo E; González, Isabel; Martín, Rosario

    2008-03-01

    A real-time PCR approach with the SYBR Green detection system has been developed for the quantitative detection of bovine tissues in food and feedstuffs. The method combines the use of bovine-specific primers, which amplify an 84-bp fragment of the mitochondrial 12S rRNA gene, and universal primers, which amplify a 140-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. The 18S rRNA primers are used as endogenous controls for the total content of PCR-amplifiable DNA in the sample. The specificity of the primers was tested against 18 animal species, including mammals, birds, and fish, as well as 6 plant species. Analysis of experimental bovine tissues-oats mixtures demonstrated the suitability of the assay for the detection of bovine DNA in mixtures containing as low as 0.1% of bovine tissues. The performance of the method is not affected by severe heat treatment (up to 133 degrees C for 20 min at 300 kPa). The reported PCR assay could be very useful for detecting bovine-derived ingredients in raw and heat-treated food and feedstuffs.

  20. Evaluation of Reference Genes for Quantitative Real-Time PCR in Songbirds

    PubMed Central

    Zinzow-Kramer, Wendy M.; Horton, Brent M.; Maney, Donna L.

    2014-01-01

    Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbird: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology. PMID:24780145

  1. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

    PubMed

    Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

    2008-04-01

    Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.

  2. Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

    PubMed

    Lebuhn, M; Effenberger, M; Garcés, G; Gronauer, A; Wilderer, P A

    2005-01-01

    In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

  3. Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree.

    PubMed

    Long, Xiangyu; He, Bin; Gao, Xinsheng; Qin, Yunxia; Yang, Jianghua; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

    2015-06-01

    In rubber tree, latex regeneration is one of the decisive factors influencing the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments.

  4. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

    PubMed Central

    Wei, Yongcheng; Liu, Qinghua; Dong, Hongyu; Zhou, Zhichun; Hao, Yanping; Chen, Xuelian; Xu, Liuyi

    2016-01-01

    Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana. PMID:26800152

  5. Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae).

    PubMed

    Shi, Caihua; Yang, Fengshan; Zhu, Xun; Du, Erxia; Yang, Yuting; Wang, Shaoli; Wu, Qingjun; Zhang, Youjun

    2016-07-07

    The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga.

  6. Identification of four squid species by quantitative real-time polymerase chain reaction.

    PubMed

    Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

    2016-02-01

    Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Meloidogyne javanica Chorismate Mutase Transcript Expression Profile Using Real-Time Quantitative RT-PCR.

    PubMed

    Painter, Janet E; Lambert, Kris N

    2003-03-01

    A developmental expression profile of the Meloidodgyne javanica esophageal gland gene chorismate mutase-1 (Mj-cm-1) could suggest when in the lifecycle of the nematode the Mj-cm-1 product is functional. This study used real-time quantitative RT-PCR to examine the variation in Mj-cm-1 transcript levels over six timepoints in the nematode lifecycle: egg, infective second-stage juveniles (Inf-J2), 2-day post-inoculation (pi), 7-day pi, 14-day pi, and adult. The Mj-cm-1 mRNA levels peaked at 2-day pi, about 100-fold above levels expressed at the egg and Inf-J2 stages. Some expression of Mj-cm-1 remained during the 7-day pi, 14-day pi, and adult stages. High transcript levels of the beta-actin control gene M. javanica Beta-actin-1 (Mj-ba-1) demonstrated the presence of cDNA at all timepoints. The peak in Mj-cm-1 transcript expression at 2-day pi as well as the previously shown esophageal gland localization of Mj-cm-1 mRNA suggest that the product of this gene may be involved early in the establishment of parasitism.

  8. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    NASA Astrophysics Data System (ADS)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85-112% (R2 = 0.962-0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

  9. Identification of Reference Genes for Real-Time Quantitative PCR Experiments in the Liverwort Marchantia polymorpha

    PubMed Central

    Dolan, Liam; Langdale, Jane A.

    2015-01-01

    Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data. PMID:25798897

  10. Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha.

    PubMed

    Saint-Marcoux, Denis; Proust, Hélène; Dolan, Liam; Langdale, Jane A

    2015-01-01

    Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.

  11. Quantifying Aotus monkey cytokines by real-time quantitative RT-PCR.

    PubMed

    Pico de Coaña, Yago; Barrero, Carlos; Cajiao, Isabela; Mosquera, Catalina; Patarroyo, Manuel Elkin; Patarroyo, Manuel Alfonso

    Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-beta and IFN-gamma). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/microl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.

  12. A new method for quantitative real-time polymerase chain reaction data analysis.

    PubMed

    Rao, Xiayu; Lai, Dejian; Huang, Xuelin

    2013-09-01

    Quantitative real-time polymerase chain reaction (qPCR) is a sensitive gene quantification method that has been extensively used in biological and biomedical fields. The currently used methods for PCR data analysis, including the threshold cycle method and linear and nonlinear model-fitting methods, all require subtracting background fluorescence. However, the removal of background fluorescence can hardly be accurate and therefore can distort results. We propose a new method, the taking-difference linear regression method, to overcome this limitation. Briefly, for each two consecutive PCR cycles, we subtract the fluorescence in the former cycle from that in the latter cycle, transforming the n cycle raw data into n-1 cycle data. Then, linear regression is applied to the natural logarithm of the transformed data. Finally, PCR amplification efficiencies and the initial DNA molecular numbers are calculated for each reaction. This taking-difference method avoids the error in subtracting an unknown background, and thus it is more accurate and reliable. This method is easy to perform, and this strategy can be extended to all current methods for PCR data analysis.

  13. Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR.

    PubMed

    Furet, Jean-Pierre; Firmesse, Olivier; Gourmelon, Michèle; Bridonneau, Chantal; Tap, Julien; Mondot, Stanislas; Doré, Joël; Corthier, Gérard

    2009-06-01

    Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups (Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.

  14. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    PubMed Central

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

  15. Quantification of Listeria monocytogenes in salads by real time quantitative PCR.

    PubMed

    Berrada, H; Soriano, J M; Picó, Y; Mañes, J

    2006-03-15

    A real time quantitative PCR (RTQ-PCR) was carried out purifying DNA extracts of Listeria monocytogenes using a High Pure Listeria Sample Preparation Kit and quantifying in a LightCycler system with hybridisation probes. A standard curve was constructed with serial dilutions. A range linear relationship, from 10 to 10(5)L. monocytogenes colony forming units (CFU), was observed between threshold cycle (Ct) and logarithmic concentration of the serial dilutions. The assay was linear in a range from 10 to 10(5)L. monocytogenes CFU and the coefficient of determination (r2) was >0.98. RTQ-PCR presented an efficiency of >85%. The accuracy of the PCR-based assay, expressed as % bias, ranged from 9% to 26% and the precision, expressed as % CV, ranged 9-22%. Intraday and interday variabilities were studied at 10(2) CFU/g and resulted in 12% and 14%, respectively. The proposed RTQ-PCR method and classical cultural methods were applied to analyse 77 salads from restaurants in Valencia (Spain). All culture positive samples were also RTQ-PCR positive.

  16. Quantitation of chemical warfare agents using the direct analysis in real time (DART) technique.

    PubMed

    Nilles, J Michael; Connell, Theresa R; Durst, H Dupont

    2009-08-15

    Direct analysis in real time (DART) is an ion source that permits rapid mass spectrometric detection of gases, liquids, and solids in open air under ambient conditions. It is a unique technology in the field of chemical weapons detectors in that it does not require a vapor pressure, does not require sample preparation, and is nondestructive to the original sample. While the DART technique has had success as a first line instrument of detection, there have been lingering doubts over the technique's quantitative reliability and reproducibility. Here, we demonstrate its capability to produce linear calibration curves (R(2) = 0.99 or better) for the nerve agents GA, GB, and VX as well as the blister agent HD. Independently prepared check standards measured against these curves typically have recovery errors less than 3%. We show the DART instrument response to be linear over roughly 3 orders of magnitude. Furthermore, this study shows that averaging as few as three measurements for each data point is sufficient to produce high quality calibration curves, thus reducing data collection time and providing quicker results.

  17. Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae).

    PubMed

    Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

    2013-01-01

    Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest.

  18. Selection of reliable reference genes for quantitative real-time RT-PCR in alfalfa.

    PubMed

    Wang, Xuemin; Fu, Yuanyuan; Ban, Liping; Wang, Zan; Feng, Guangyan; Li, Jun; Gao, Hongwen

    2015-01-01

    Real-time quantitative RT-PCR (qRT-PCR) is the most commonly used method for accurately detecting gene expression patterns. As part of qRT-PCR analysis, normalization of the data requires internal control gene(s) that display uniform expression under different biological conditions. However, no invariable internal control gene exists, and therefore more than one reference gene is needed to normalize RT-PCR results. In this study, we assessed the expression of eight candidate internal control genes, namely 18S ribosomal RNA (18S rRNA), elongation factor-1alpha, β-Actin, E2 ubiquitin-conjugating enzyme, β-Tubulin (TUB), ACTIN2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Msc27 of unknown function, in a diverse set of 16 alfalfa (Medicago sativa) samples representing different tissues and abiotic stress challenges, using geNorm and BestKeeper software. The results revealed that the eight candidate genes are inconsistently expressed under different experimental conditions. Msc27 and 18S rRNA are suitable reference genes for comparing different tissue types. Under different abscisic acid and NaCl conditions, three reference genes are necessary. Finally, GAPDH, TUB and β-Actin are unsuitable for normalization of qRT-PCR data under these given conditions in alfalfa. The relative expression level of MsWRKY33 was analyzed using selected reference genes. These results provide an experimental guideline for future research on gene expression in alfalfa using qRT-PCR.

  19. GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

    PubMed Central

    Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

    2011-01-01

    The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch. PMID:21917859

  20. A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

    USGS Publications Warehouse

    Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

    2015-01-01

    Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

  1. Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR.

    PubMed

    Hierro, Núria; Esteve-Zarzoso, Braulio; Mas, Albert; Guillamón, Jose M

    2007-12-01

    Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.

  2. Identification of five highly priced tuna species by quantitative real-time polymerase chain reaction.

    PubMed

    Liu, Shasha; Xu, Kunhua; Wu, Zhigang; Xie, Xiao; Feng, Junli

    2016-09-01

    Tunas are economically important fishery worldwide, and are often used for commercial processed production. For effective fishery management and protection of consumers' rights, it is important to develop a molecular method to identify species in canned tuna products rapidly and reliably. Here, we have developed a duplex quantitative real-time PCR (qPCR) for identification of five highly priced tuna species (Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis) from processed as well as fresh fish. After amplification and sequencing of seven genetic markers commonly used for species identification, 16S rDNA and control region (CR) of mitochondrial DNA were selected as the reference gene markers for genus Thunnus and tuna species identification, respectively. Subsequently, a 73 bp fragment of 16S rDNA and 85-99 bp fragment of CR were simultaneously amplified from each target species by qPCR. The qPCR efficiency of each reaction was calculated according to the standard curves, and the method was validated by amplification DNA extracted from single or mixed tuna specimen. The developed duplex qPCR system was applied to authenticate species of 14 commercial tuna products successfully, which demonstrated it was really a useful and academic technique to identify highly priced tuna species.

  3. A quantitative real-time approach for discriminating apoptosis and necrosis

    PubMed Central

    Lekshmi, Asha; Varadarajan, Shankara Narayanan; Lupitha, Santhik Subhasingh; Indira, Deepa; Mathew, Krupa Ann; Chandrasekharan Nair, Aneesh; Nair, Mydhily; Prasad, Tilak; Sekar, Hari; Gopalakrishnan, Anurup Kochucherukkan; Murali, Abitha; Santhoshkumar, Thankayyan Retnabai

    2017-01-01

    Apoptosis and necrosis are the two major forms of cell death mechanisms. Both forms of cell death are involved in several physiological and pathological conditions and also in the elimination of cancer cells following successful chemotherapy. Large number of cellular and biochemical assays have evolved to determine apoptosis or necrosis for qualitative and quantitative purposes. A closer analysis of the assays and their performance reveal the difficulty in using any of these methods as a confirmatory approach, owing to the secondary induction of necrosis in apoptotic cells. This highlights the essential requirement of an approach with a real-time analysis capability for discriminating the two forms of cell death. This paper describes a sensitive live cell-based method for distinguishing apoptosis and necrosis at single-cell level. The method uses cancer cells stably expressing genetically encoded FRET-based active caspase detection probe and DsRed fluorescent protein targeted to mitochondria. Caspase activation is visualized by loss of FRET upon cleavage of the FRET probe, while retention of mitochondrial fluorescence and loss of FRET probe before its cleavage confirms necrosis. The absence of cleavage as well as the retention of mitochondrial fluorescence indicates live cells. The method described here forms an extremely sensitive tool to visualize and quantify apoptosis and necrosis, which is adaptable for diverse microscopic, flow cytometric techniques and high-throughput imaging platforms with potential application in diverse areas of cell biology and oncology drug screening. PMID:28179996

  4. Estimation of transgene copy number in transformed citrus plants by quantitative multiplex real-time PCR.

    PubMed

    Omar, Ahmad A; Dekkers, Marty G H; Graham, James H; Grosser, Jude W

    2008-01-01

    Quantitative real-time PCR (qRT-PCR) was adapted to estimate transgene copy number of the rice Xa21 gene in transgenic citrus plants. This system used TaqMan qRT-PCR and the endogenous citrus gene encoding for lipid transfer protein (LTP). Transgenic "Hamlin" sweet orange plants were generated using two different protoplast-GFP transformation systems: cotransformation and single plasmid transformation. A dilution series of genomic DNA from one of the transgenic lines was used to generate a standard curve for the endogenous LTP and the transgene Xa21. This standard curve was used for relative quantification of the endogenous gene and the transgene. Copy numbers of the transgene Xa21 detected from qRT-PCR analysis correlated with that from Southern blot analysis (r = 0.834). Thus, qRT-PCR is an efficient means of estimating copy number in transgenic citrus plants. This analysis can be performed at much earlier stages of transgenic plant development than southern blot analysis, which expedites investigation of transgenes in slow-growing woody plants.

  5. Microscopy, culture, and quantitative real-time PCR examination confirm internalization of mycobacteria in plants.

    PubMed

    Kaevska, M; Lvoncik, S; Slana, I; Kulich, P; Kralik, P

    2014-07-01

    The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.

  6. Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae)

    PubMed Central

    Shi, Caihua; Yang, Fengshan; Zhu, Xun; Du, Erxia; Yang, Yuting; Wang, Shaoli; Wu, Qingjun; Zhang, Youjun

    2016-01-01

    The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga. PMID:27399679

  7. Development of a Quantitative Real-Time PCR Assay for Detection of Mycoplasma genitalium

    PubMed Central

    Svenstrup, Helle Friis; Jensen, Jørgen Skov; Björnelius, Eva; Lidbrink, Peter; Birkelund, Svend; Christiansen, Gunna

    2005-01-01

    Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/μl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods. PMID:16000423

  8. Virological Diagnosis of Herpes Simplex Virus 1 Esophagitis by Quantitative Real-Time PCR Assay

    PubMed Central

    Jazeron, Jean-François; Barbe, Coralie; Frobert, Emilie; Renois, Fanny; Talmud, Déborah; Brixi-Benmansour, Hedia; Brodard, Véronique; Andréoletti, Laurent; Diebold, Marie-Danièle

    2012-01-01

    Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the “gold standard.” From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 106 ± 1.1 × 108) than in histopathologically negative herpetic esophagitis (median = 3.1 × 103 ± 6.2 × 103) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 104 copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain. PMID:22170921

  9. Quantitative detection of Vibrio cholera toxin by real-time and dynamic cytotoxicity monitoring.

    PubMed

    Jin, Dazhi; Luo, Yun; Zheng, Min; Li, Haijing; Zhang, Jing; Stampfl, Melinda; Xu, Xiao; Ding, Gangqiang; Zhang, Yanjun; Tang, Yi-Wei

    2013-12-01

    We report here the quantitative detection of Vibrio cholerae toxin (CT) in isolates and stool specimens by dynamic monitoring of the full course of CT-mediated cytotoxicity in a real-time cell analysis (RTCA) system. Four cell lines, including Y-1 mouse adrenal tumor cells, Chinese hamster ovary (CHO) cells, small intestine epithelial (FHs74Int) cells, and mouse adrenal gland (PC12-Adh) cells, were evaluated for their suitability for CT-induced cytotoxicity testing. Among them, the Y-1 line was demonstrated to be the most sensitive for CT-mediated cytotoxicity, with limits of detection of 7.0 pg/ml for purified CT and 0.11 ng/ml for spiked CT in pooled negative stool specimens. No CT-mediated cytotoxicity was observed for nontoxigenic V. cholerae, non-V. cholerae species, or non-V. cholerae enterotoxins. The CT-RTCA assay was further validated with 100 stool specimens consecutively collected from patients with diarrhea and 200 V. cholerae isolates recovered from patients and the environment, in comparison to a reference using three detection methods. The CT-RTCA assay had sensitivities and specificities of 97.5% and 100.0%, respectively, for V. cholerae isolates and 90.0% and 97.2% for stool specimens. For stool specimens spiked with CT concentrations ranging from 3.5 pg/ml to 1.8 ng/ml, the inoculation-to-detection time was 1.12 ± 0.38 h, and the values were inversely correlated with CT concentrations (ρ = -1; P = 0.01). The results indicate that the CT-RTCA assay with the Y-1 cell line provides a rapid and sensitive tool for the quantitative detection of CT activities in clinical specimens.

  10. [Comparison of direct immune-fluorescent assay and real-time quantitative PCR in detecting the Hantavirus].

    PubMed

    Yu, Peng-bo; Li, Shen; Wei, Jing; Ma, Chang-an; Lu, Xiao-ling; DU, Shui-quan; Guan, Lu-yuan; Zheng, Yuan; Dong, Jian-hua; Ma, Chao-feng; Wang, Jing-jun

    2013-04-01

    To compare the differences between the direct immuno-fluorescent assay (DFA) and real-time quantitative PCR in detecting the Hantavirus (HV) in rat lungs. From April to October in 2012, a total of 479 rats were caught by mouse-trap in residential or wild areas in Huxian, Jingyang, and Meixian of Shaanxi province, where haemorrhagic fever with renal syndrome (HFRS) was highly prevalent. The rats were dissected to take the two lungs, one was frozen and applied immuno-fluorescent assay to detect HV antigen while the other one was extracted its RNA and detected HV nucleic acid by real-time quantitative PCR. Then we compared the positive rate of the two methods. Out of the 479 rats, 105 were caught from residential areas and the other 374 were caught from wild areas. Among the 105 rats caught from residential areas, no HV were detected out neither by DFA nor by real-time quantitative PCR. Among the 374 wild rats, 13.1% (49/374) were detected HV positive by DFA and 14.7% (55/374) were detected HV positive by real-time quantitative PCR. The difference showed no statistical significance (χ(2) = 0.402, P = 0.526). When detecting each lung sample, the HV positive rate was 10.2% (49/479) under the detection by DFA while the HV positive rate was 11.5% (55/479) under the detection by real-time quantitative PCR. The difference had no statistical significance (χ(2) = 1.286, P = 0.257) and the consistency coefficient was 68.2% under the paired chi-square test analysis, which showed high consistency (u = 11.759, P < 0.05). The sensitivity of real-time quantitative PCR to detect HV was 77.6% (38/49) comparing with DFA as standard, and the specificity was 96.1% (413/430). Out of the 9 suspected HV positive sample detected by DFA, 6 were confirmed positive by real-time quantitative PCR and 3 were denied. Compared with the DFA, real-time quantitative PCR could also be used to detect the infection of HV in rats, and the result might be more stable.

  11. Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

    PubMed

    Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

    2014-01-01

    Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

  12. Expression of nisin genes in cheese--a quantitative real-time polymerase chain reaction approach.

    PubMed

    Trmčić, A; Monnet, C; Rogelj, I; Bogovič Matijašić, B

    2011-01-01

    The role of bacteriocins in different environments has not been thoroughly explained, mainly because of the difficulties related to the detection of their production. Nisin, an antimicrobial peptide produced by Lactococcus lactis has a long history of safe use in food products and has been studied from many aspects of genetics, biosynthesis, immunity, regulation, and mode of action. Still, some aspects concerning the dynamics of nisin gene expression remain unknown, especially in complex media like cheese. The main objective of the present study was to quantify in a cheese-like medium the expression of nisin genes in L. lactis M78, a well-characterized nisin A producer isolated from raw milk. The expression of all 11 genes involved in nisin biosynthesis was evaluated during cheese production by real-time reverse transcription-PCR. Total RNA was extracted from cheeses using a direct extraction method without prior separation of microbial cells. The M78 strain grew well in experimental cheeses, producing detectable amounts of nisin after 4 h of fermentation. The presence of nisin as an activator modified both the expression of nisin genes and the accumulation of active nisin. Four groups could be distinguished based on gene expression as a function of time: nisA, nisFEG, nisRK and nisBTCIP. Based on nisin-producing strain growth, nisin activity, function of nisin genes, and their location, correlations were established that contribute to the explanation of regulation of nisin biosynthesis and immunity. This study is the first in which the evolution of bacteriocin gene transcripts has been quantified rigorously in a cheese-like medium.

  13. Comparison of Quantitation of Cytomegalovirus DNA by Real-Time PCR in Whole Blood with the Cytomegalovirus Antigenemia Assay

    PubMed Central

    Kwon, Seonhee; Jung, Bo Kyeung; Ko, Sun-Young; Lee, Chang Kyu

    2015-01-01

    Background Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. Method 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. Results The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. Conclusions Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients. PMID:25553288

  14. Development of universal SYBR Green real-time RT-PCR for the rapid detection and quantitation of bovine and porcine toroviruses.

    PubMed

    Hosmillo, Myra D T; Jeong, Young-Ju; Kim, Hyun-Jeong; Collantes, Therese Marie; Alfajaro, Mia Madel; Park, Jun-Gyu; Kim, Ha-Hyun; Kwon, Hyung-Jun; Park, Su-Jin; Kang, Mun-Il; Park, Sang-Ik; Cho, Kyoung-Oh

    2010-09-01

    Toroviruses (ToVs) are a group of emerging viruses that cause gastroenteritis in domestic animals and humans. Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Using BToV and PToV RNA standards generated by in vitro transcription, the detection limit of the SYBR Green real-time RT-PCR assay was 2.54 x 10(2) BToV and 2.17 x 10(3) PToV copies/reaction (correlation coefficiency=0.99 and 0.97, respectively), whereas those of RT-PCR and nested PCR were 2.54 x 10(5) and 2.54 x 10(4) (BToV) and 2.17 x 10(7) and 2.17 x 10(5) (PToV) cRNA viral copies/reaction, respectively. Archived diarrhea specimens of calves (n=121) and piglets (n=86) were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR, 1 (0.8%) bovine and 7 (8.1%) porcine samples tested positive to BToV and PToV, respectively. With nested PCR, 13 (10.7%) bovine and 17 (19.8%) porcine samples tested positive. SYBR Green real-time RT-PCR assay detected BToV and PToV in 22 of 121 (18.2%) bovine and 31 of 86 (36.0%) porcine samples. These results indicate that SYBR Green real-time RT-PCR (P<0.05) is a more sensitive assay, which can be reproduced as a reliable, sensitive, and rapid tool for the detection and quantitation of toroviruses.

  15. Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in Sesamia inferens (Lepidoptera: Noctuidae)

    PubMed Central

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (−8, −6, −4, −2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens. PMID:25585250

  16. Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Mulvey, Christine S.; Sherwood, Carly A.; Bigio, Irving J.

    2009-11-01

    Apoptosis-programmed cell death-is a cellular process exhibiting distinct biochemical and morphological changes. An understanding of the early morphological changes that a cell undergoes during apoptosis can provide the opportunity to monitor apoptosis in tissue, yielding diagnostic and prognostic information. There is avid interest regarding the involvement of apoptosis in cancer. The initial response of a tumor to successful cancer treatment is often massive apoptosis. Current apoptosis detection methods require cell culture disruption. Our aim is to develop a nondisruptive optical method to monitor apoptosis in living cells and tissues. This would allow for real-time evaluation of apoptotic progression of the same cell culture over time without alteration. Elastic scattering spectroscopy (ESS) is used to monitor changes in light-scattering properties of cells in vitro due to apoptotic morphology changes. We develop a simple instrument capable of wavelength-resolved ESS measurements from cell cultures in the backward direction. Using Mie theory, we also develop an algorithm that extracts the size distribution of scatterers in the sample. The instrument and algorithm are validated with microsphere suspensions. For cell studies, Chinese hamster ovary (CHO) cells are cultured to confluence on plates and are rendered apoptotic with staurosporine. Backscattering measurements are performed on pairs of treated and control samples at a sequence of times up to 6-h post-treatment. Initial results indicate that ESS is capable of discriminating between treated and control samples as early as 10- to 15-min post-treatment, much earlier than is sensed by standard assays for apoptosis. Extracted size distributions from treated and control samples show a decrease in Rayleigh and 150-nm scatterers, relative to control samples, with a corresponding increase in 200-nm particles. Work continues to correlate these size distributions with underlying morphology. To our knowledge, this

  17. The quantification of spermatozoa by real-time quantitative PCR, spectrophotometry, and spermatophore cap size.

    PubMed

    Doyle, Jacqueline M; McCormick, Cory R; DeWoody, J Andrew

    2011-01-01

    Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity.

  18. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

    PubMed Central

    2011-01-01

    Background Quantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. Results We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight) exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer). These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. Conclusions The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty. PMID:21501492

  19. Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies

    PubMed Central

    Wong, Samson S. Y.; Poon, Rosana W. S.; Chau, Sandy; Wong, Sally C. Y.; To, Kelvin K. W.; Cheng, Vincent C. C.; Fung, Kitty S. C.

    2015-01-01

    Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. PMID:25903566

  20. Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

    PubMed

    Pessoa-E-Silva, Rômulo; Mendonça Trajano-Silva, Lays Adrianne; Lopes da Silva, Maria Almerice; da Cunha Gonçalves-de-Albuquerque, Suênia; de Goes, Tayná Correia; Silva de Morais, Rayana Carla; Lopes de Melo, Fábio; de Paiva-Cavalcanti, Milena

    2016-12-01

    The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per μL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies.

    PubMed

    Wong, Samson S Y; Poon, Rosana W S; Chau, Sandy; Wong, Sally C Y; To, Kelvin K W; Cheng, Vincent C C; Fung, Kitty S C; Yuen, K Y

    2015-07-01

    Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

    PubMed Central

    2012-01-01

    Background Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species. PMID:23146128

  3. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

    PubMed

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  4. The use of reverse iontophoresis based surface plasmon resonance for the development of a noninvasive real time transdermal biomarker sensor

    NASA Astrophysics Data System (ADS)

    Gupta, Niraj K.; Hwang, Yongsoon; Cameron, Brent D.

    2016-03-01

    Recent developments in the identification of biomarkers offer a potential means to facilitate early disease detection, gauge treatment in drug therapy clinical trials, and to assess the impact of fatigue and/or stress as related to human physical and cognitive performance. For practical implementation, however, real-time sensing and quantification of such physiological biomarkers is preferred. Some key aspects in this process are continuous sample collection and real time detection. Traditionally, blood is considered the gold standard for samples but frequent phlebotomy is painful and inconvenient. Other sources like saliva and passive sweat cannot be precisely controlled and are affected by other limitations. Some of these can be addressed by reverse iontophoresis which is a noninvasive technique capable of facilitating controlled transport of biomolecules up to 20kDa in size across the skin barrier by passing a low level current between two dermal electrodes. The samples collected at the electrode site can then be monitored at site or transported via a microfluidic channel towards a sensor. In the case reported here, the sensor is based on surface plasmon resonance (SPR), which is a label free, real time, and highly sensitive optical sensing technique. The real time SPR detection of targeted biomarkers is then achieved through the use of aptamer surface modification. In this experiment, extraction and detection of orexin A, a stress related biomarker, is used for demonstration purposes.

  5. Detection of Avian bornavirus in multiple tissues of infected psittacine birds using real-time reverse transcription polymerase chain reaction.

    PubMed

    Delnatte, Pauline; Mak, Matthew; Ojkic, Davor; Raghav, Raj; DeLay, Josepha; Smith, Dale A

    2014-03-01

    Avian bornavirus (ABV), the cause of proventricular dilation disease in psittacine birds, has been detected in multiple tissues of infected birds using immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR). In the current study, real-time RT-PCR, using primers targeting the ABV matrix gene, was used to detect ABV in 146 tissues from 7 ABV-infected psittacine birds. Eighty-six percent of the samples tested positive, with crossing point values ranging from 13.82 to 37.82 and a mean of 22.3. These results were compared to the findings of a previous study using gel-based RT-PCR and IHC on the same samples. The agreement between the 2 RT-PCR techniques was 91%; when tests disagreed it was because samples were negative using gel-based RT-PCR but positive on real-time RT-PCR. Agreement with IHC was 77%; 16 out of 74 samples were negative using IHC but positive on real-time RT-PCR. The results suggest that real-time RT-PCR is a more sensitive technique than gel-based RT-PCR and IHC to detect ABV in tissues. The tissues that were ranked most frequently as having a high amount of viral RNA were proventriculus, kidney, colon, cerebrum, and cerebellum. Skeletal muscle, on the other hand, was found to have a consistently low amount of viral RNA.

  6. A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

    PubMed

    Scherrer, Simone; Frei, Daniel; Wittenbrink, Max Michael

    2016-12-01

    Progressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs. In comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR. The approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.

  7. Comparison of Sensitivity and Quantitation between Microbead Dielectrophoresis-Based DNA Detection and Real-Time PCR.

    PubMed

    Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya

    2017-09-30

    In this study, we describe a microbead-based method using dielectrophoresis (DEP) for the fast detection of DNA amplified by polymerase chain reaction (PCR). This electrical method measures the change in impedance caused by DEP-trapped microbeads to which biotinylated target DNA molecules are chemically attached. Using this method, measurements can be obtained within 20 min. Currently, real-time PCR is among the most sensitive methods available for the detection of target DNA, and is often used in the diagnosis of infectious diseases. We therefore compared the quantitation and sensitivity achieved by our method to those achieved with real-time PCR. We found that the microbead DEP-based method exhibited the same detection limit as real-time PCR, although its quantitative detection range was slightly narrower at 10-10⁵ copies/reaction compared with 10-10⁷ copies/reaction for real-time PCR. Whereas real-time PCR requires expensive and complex instruments, as well as expertise in primer design and experimental principles, our novel method is simple to use, inexpensive, and rapid. This method could potentially detect viral and other DNAs efficiently in combination with conventional PCR.

  8. Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR

    PubMed Central

    Fakhari, Farnaz D.; Dittmer, Dirk P.

    2002-01-01

    The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome. PMID:12021355

  9. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2013-12-01

    Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.

  10. Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR.

    PubMed

    Xu, Yuanyuan; Zhu, Xianwen; Gong, Yiqin; Xu, Liang; Wang, Yan; Liu, Liwang

    2012-08-03

    Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples.

    PubMed

    Nicklas, Janice A; Buel, Eric

    2003-09-01

    Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming, and labor intensive. We have previously described a method for quantitation of human DNA based on PCR amplification of a repetitive Alu sequence that uses a fluorescence plate reader. This manuscript describes and validates a variation of this assay using real-time PCR and SYBR Green I for quantitation. The advantages of the real-time assay over the plate reader assay are: reduced hands-on time, lower assay cost, and a greater dynamic range. The main disadvantage is the cost of the real-time instrument. However, for those forensic laboratories with access to a real-time instrument, this Alu-based assay has a dynamic range of 16 ng to 1 pg, is sensitive, specific, fast, quantitative, and uses only 2 microL of sample.

  12. EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

    EPA Science Inventory

    Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

  13. EVALUATION OF RAPID DNA EXTRACTION PROCEDURES FOR THE QUANTITATIVE DETECTION OF FUNGAL CELLS USING REAL TIME PCR ANALYSIS

    EPA Science Inventory

    The ease and rapidity of quantitative DNA sequence detection by real-time PCR instruments promises to make their use increasingly common for the microbial analysis many different types of environmental samples. To fully exploit the capabilities of these instruments, correspondin...

  14. A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

    EPA Science Inventory

    In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

  15. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

    EPA Science Inventory

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

  16. Duplex TaqMan real-time PCR assay for quantitative detection of Pantoea stewartii subsp. stewartii and Stenocarpella maydis

    USDA-ARS?s Scientific Manuscript database

    A new TaqMan real-time PCR assay was developed for the simultaneous quantitative detection of two seedborne maize pathogens in a single assay. Pantoea stewartii subsp. stewartii (Pnss) (syn. Erwinia stewartii) is the causal agent of Stewart's bacterial wilt and leaf blight of maize. Stewart's wilt i...

  17. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

    EPA Science Inventory

    There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

  18. Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary by Real-Time Quantitative PCR

    EPA Science Inventory

    The Salt Creek watershed in northwest Indiana drains into Lake Michigan near several heavily used recreational beaches. This study aimed to investigate the levels of fecal indicator bacteria, enterococci and Bacteroidales, in Salt Creek using real-time quantitative PCR (qPCR) an...

  19. A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

    EPA Science Inventory

    Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

  20. EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

    EPA Science Inventory

    Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

  1. Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

    USDA-ARS?s Scientific Manuscript database

    The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

  2. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

    EPA Science Inventory

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

  3. Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary by Real-Time Quantitative PCR

    EPA Science Inventory

    The Salt Creek watershed in northwest Indiana drains into Lake Michigan near several heavily used recreational beaches. This study aimed to investigate the levels of fecal indicator bacteria, enterococci and Bacteroidales, in Salt Creek using real-time quantitative PCR (qPCR) an...

  4. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

    EPA Science Inventory

    There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

  5. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

    USDA-ARS?s Scientific Manuscript database

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster have been found to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. The United States Environmental Protection Agency is planning to conduct a ...

  6. Decay Of Bacterial Pathogen, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure Amended Soils

    EPA Science Inventory

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria, and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manre-amended agricultural soils. Known concentrations of transformed green fluore...

  7. Decay Of Bacterial Pathogens, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure-Amended Soils

    EPA Science Inventory

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green...

  8. Validation and standardization of gene expression data for microarray and real time quantitative PCR using universal external RNA controls

    USDA-ARS?s Scientific Manuscript database

    This presentation will introduce newly developed universal external ribonucleic acid (RNA) controls and their applications on different platforms of microarray and quantitative real time polymerase chain reaction (qRT-PCR) including SYBR Green® and TaqMan® probe-based chemistries. Data obtained fro...

  9. Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method ...

  10. Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution - Poster

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method p...

  11. EVALUATION OF RAPID DNA EXTRACTION PROCEDURES FOR THE QUANTITATIVE DETECTION OF FUNGAL CELLS USING REAL TIME PCR ANALYSIS

    EPA Science Inventory

    The ease and rapidity of quantitative DNA sequence detection by real-time PCR instruments promises to make their use increasingly common for the microbial analysis many different types of environmental samples. To fully exploit the capabilities of these instruments, correspondin...

  12. A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

    EPA Science Inventory

    In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

  13. A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

    EPA Science Inventory

    Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

  14. A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR

    USDA-ARS?s Scientific Manuscript database

    Microarray analysis and quantitative real-time RT-PCR are the major high-throughput techniques that are used to study transcript profiles. One of the major limitations in these technologies is the isolation maximum yield of highly-pure RNA from plant tissues rich in complex polysaccharides, polyphen...

  15. Decay Of Bacterial Pathogen, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure Amended Soils

    EPA Science Inventory

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria, and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manre-amended agricultural soils. Known concentrations of transformed green fluore...

  16. Decay Of Bacterial Pathogens, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure-Amended Soils

    EPA Science Inventory

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green...

  17. An integrated real-time diagnostic concept using expert systems, qualitative reasoning and quantitative analysis

    SciTech Connect

    Edwards, R.M.; Lee, K.Y.; Kumara, S.; Levine, S.H.

    1989-01-01

    An approach for an integrated real-time diagnostic system is being developed for inclusion as an integral part of a power plant automatic control system. In order to participate in control decisions and automatic closed loop operation, the diagnostic system must operate in real-time. Thus far, an expert system with real-time capabilities has been developed and installed on a subsystem at the Experimental Breeder Reactor (EBR-II) in Idaho, USA. Real-time simulation testing of advanced power plant concepts at the Pennsylvania State University has been developed and was used to support the expert system development and installation at EBR-II. Recently, the US National Science Foundation (NSF) and the US Department of Energy (DOE) have funded a Penn State research program to further enhance application of real-time diagnostic systems by pursuing implementation in a distributed power plant computer system including microprocessor based controllers. This paper summarizes past, current, planned, and possible future approaches to power plant diagnostic systems research at Penn State. 34 refs., 9 figs.

  18. Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.

    PubMed

    Takahashi, Hajime; Saito, Rumi; Miya, Satoko; Tanaka, Yuichiro; Miyamura, Natsumi; Kuda, Takashi; Kimura, Bon

    2017-04-04

    The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A <1log difference between the real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r(2)=0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Quantitative detection of hazelnut (Corylus avellana) in cookies: ELISA versus real-time PCR.

    PubMed

    Platteau, Céline; De Loose, Marc; De Meulenaer, Bruno; Taverniers, Isabel

    2011-11-09

    Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.

  20. Comparison of real-time PCR, reverse transcriptase real-time PCR, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce.

    PubMed

    Zhang, Guodong; Brown, Eric W; González-Escalona, Narjol

    2011-09-01

    Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (10(5) and <10(1) CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities.

  1. Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ▿ †

    PubMed Central

    Zhang, Guodong; Brown, Eric W.; González-Escalona, Narjol

    2011-01-01

    Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and <101 CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

  2. Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

    PubMed

    Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

    2017-08-15

    Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R(2) value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

  3. Comparison of TaqMan and Epoch Dark Quenchers during real-time reverse transcription PCR.

    PubMed

    Daum, Luke T; Ye, Keying; Chambers, James P; Santiago, Jose; Hickman, John R; Barnes, William J; Kruzelock, Russell P; Atchley, Daniel H

    2004-06-01

    Several biotechnology companies have recently introduced novel quencher fluors for use with dual-labeled fluorogenic hydrolysis probes. The Epoch Dark Quencher trade mark fluorochrome consists of a non-fluorescent moiety capable of absorption at higher wavelengths (400-650 nm). The aim of this study was to: (1) evaluate the feasibility of using Epoch Dark Quencher fluorochromes in real-time PCR pathogen detection assays that were previously optimized with TaqMan (TAMRA) quenching fluors, and (2) compare the sensitivity based on cycle threshold (CT) between probes containing either TaqMan or Epoch Dark Quencher fluors. Our data indicate Epoch Dark Quencher probes can be used in place of TaqMan probes and their performance was not better than traditional TaqMan (TAMRA) quenchers. Marginal differences observed between quenching fluorochromes may arise from concentration differences during probe synthesis.

  4. Blood purine measurements as a rapid real-time indicator of reversible brain ischaemia.

    PubMed

    Tian, Faming; Bibi, Fakhra; Dale, Nicholas; Imray, Christopher H E

    2017-08-12

    To preserve the disequilibrium between ATP and ADP necessary to drive cellular metabolism, enzymatic pathways rapidly convert ADP to adenosine and the downstream purines inosine and hypoxanthine. During ischaemia, these same pathways result in the production of purines. We performed a prospective observational study to test whether purine levels in arterial blood might correlate with brain ischaemia. We made real-time perioperative measurements, via microelectrode biosensors, of the purine levels in untreated arterial blood from 18 patients undergoing regional anaesthetic carotid endarterectomy. Pre-operatively, the median purine level was 2.4 μM (95% CI 1.3-4.0 μM); during the cross-clamp phase, the purines rose to 6.7 μM (95% CI 4.7-11.5 μM) and fell back to 1.9 μM (95% CI 1.4-2.7 μM) in recovery. Three patients became unconscious during carotid clamping, necessitating insertion of a temporary carotid shunt to restore cerebral blood flow. In these, the pre-operative median purine level was 5.4 μM (range 4.7-6.1 μM), on clamping, 9.6 μM (range 9.4-16.1 μM); during shunting, purines fell to below the pre-operative level (1.4 μM, range 0.4-2.9 μM) and in recovery 1.8 μM (range 1.8-2.6 μM). Our results suggest that blood purines may be a sensitive real-time and rapidly produced indicator of brain ischaemia, even when there is no accompanying neurological obtundation.

  5. Investigating reference genes for quantitative real-time PCR analysis across four chicken tissues.

    PubMed

    Bagés, S; Estany, J; Tor, M; Pena, R N

    2015-04-25

    Accurate normalization of data is required to correct for different efficiencies and errors during the processing of samples in reverse transcription PCR analysis. The chicken is one of the main livestock species and its genome was one of the first reported and used in large scale transcriptomic analysis. Despite this, the chicken has not been investigated regarding the identification of reference genes suitable for the quantitative PCR analysis of growth and fattening genes. In this study, five candidate reference genes (B2M, RPL32, SDHA, TBP and YWHAZ) were evaluated to determine the most stable internal reference for quantitative PCR normalization in the two main commercial muscles (pectoralis major (breast) and biceps femoris (thigh)), liver and abdominal fat. Four statistical methods (geNorm, NormFinder, CV and BestKeeper) were used in the evaluation of the most suitable combination of reference genes. Additionally, a comprehensive ranking was established with the RefFinder tool. This analysis identified YWHAZ and TBP as the recommended combination for the analysis of biceps femoris and liver, YWHAZ and RPL32 for pectoralis major and RPL32 and B2M for abdominal fat and across-tissue studies. The final ranking for each tool changed slightly but overall the results, and most particularly the ability to discard the least robust candidates, were consistent between tools. The selection and number of reference genes were validated using SCD, a target gene related to fat metabolism. Overall, the results can be directly used to quantitate target gene expression in different tissues or in validation studies from larger transcriptomic experiments. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Avian influenza virus detection and quantitation by real-time RT-PCR

    USDA-ARS?s Scientific Manuscript database

    Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

  7. [PCR "real time" to analyze the quantitative and qualitative relations microbiota of periodontal pockets].

    PubMed

    Zorina, O A; Kulakov, A A; Boriskina, O A; Rebrikov, D V

    2011-01-01

    The introduction of a broad medical practice PCR "real time" is just beginning and dentistry is no exception. Modern molecular genetic methods provide numerous opportunities for diagnosis, assessment and prediction in patients with inflammatory periodontal diseases. Early and accurate diagnosis can allow in the future reduce the incidence of periodontitis and the progression of its course.

  8. Detection of Human Cytomegalovirus DNA by Real-Time Quantitative PCR

    PubMed Central

    Nitsche, Andreas; Steuer, Nina; Schmidt, Christian Andreas; Landt, Olfert; Ellerbrok, Heinz; Pauli, Georg; Siegert, Wolfgang

    2000-01-01

    A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients. PMID:10878073

  9. An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples.

    PubMed

    Nicklas, Janice A; Buel, Eric

    2005-09-01

    The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).

  10. A new, multiplex, quantitative real-time polymerase chain reaction system for nucleic acid detection and quantification.

    PubMed

    Liang, Fang; Arora, Neetika; Zhang, Kang Liang; Yeh, David Che Cheng; Lai, Richard; Pearson, Darnley; Barnett, Graeme; Whiley, David; Sloots, Theo; Corrie, Simon R; Barnard, Ross T

    2013-01-01

    Quantitative real-time polymerase chain reaction (qPCR) has emerged as a powerful investigative and diagnostic tool with potential to generate accurate and reproducible results. qPCR can be designed to fulfil the four key aspects required for the detection of nucleic acids: simplicity, speed, sensitivity, and specificity. This chapter reports the development of a novel real-time multiplex quantitative PCR technology, dubbed PrimRglo™, with a potential for high-degree multiplexing. It combines the capacity to simultaneously detect many viruses, bacteria, or nucleic acids, in a single reaction tube, with the ability to quantitate viral or bacterial load. The system utilizes oligonucleotide-tagged PCR primers, along with complementary fluorophore-labelled and quencher-labelled oligonucleotides. The analytic sensitivity of PrimRglo technology was compared with the widely used Taqman(®) and SYBR green detection systems.

  11. Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods.

    PubMed

    Vaïtilingom, M; Pijnenburg, H; Gendre, F; Brignon, P

    1999-12-01

    A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.

  12. Real-Time Reverse-Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Rift Valley Fever Virus▿

    PubMed Central

    Peyrefitte, Christophe N.; Boubis, Laetitia; Coudrier, Daniel; Bouloy, Michèle; Grandadam, Marc; Tolou, Hugues J.; Plumet, Sébastien

    2008-01-01

    The development and validation of a one-step, single-tube, real-time accelerated reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the L RNA segment of Rift Valley fever virus (RVFV) are described. The assay was performed at a constant temperature (63°C), with a real-time follow-up using a LightCycler and a double-stranded-DNA-intercalating fluorochrome. The assay is highly sensitive and comparable to real-time RT-PCR, with a detection limit of ∼10 RNA copies per assay. However, the RT-LAMP assay is much faster than traditional RT-PCR and generates results in <30 min for most diluted samples. The specificity of the primers was established using other, related arboviruses as well as virus-containing and virus-free sera. The RT-LAMP assay reported here is thus a valuable tool for the rapid detection of RVFV in field diagnostic laboratories. PMID:18799705

  13. Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food.

    PubMed

    Yoon, So-Yeon; Chung, Gyung Tae; Kang, Do-Hyun; Ryu, Chunsun; Yoo, Cheon-Kwon; Seong, Won-Keun

    2005-01-01

    The TaqMan real-time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence-specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10-fold dilution series of DNA and spores . The DNA and spores were detected up to level of 0.1 ng/ml and 10(2)spores/ml, respectively. Spore spiked food sample preparation prior to the real-time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 10(2) spores/ml for spiked sausage slurry, and 10(3) spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 10(2) spores/ml in both sausage and canned corn. Therefore the real-time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real-time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.

  14. System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation

    PubMed Central

    Spiess, Andrej-Nikolai; Rödiger, Stefan; Burdukiewicz, Michał; Volksdorf, Thomas; Tellinghuisen, Joel

    2016-01-01

    Real-time quantitative polymerase chain reaction (qPCR) data are found to display periodic patterns in the fluorescence intensity as a function of sample number for fixed cycle number. This behavior is seen for technical replicate datasets recorded on several different commercial instruments; it occurs in the baseline region and typically increases with increasing cycle number in the growth and plateau regions. Autocorrelation analysis reveals periodicities of 12 for 96-well systems and 24 for a 384-well system, indicating a correlation with block architecture. Passive dye experiments show that the effect may be from optical detector bias. Importantly, the signal periodicity manifests as periodicity in quantification cycle (Cq) values when these are estimated by the widely applied fixed threshold approach, but not when scale-insensitive markers like first- and second-derivative maxima are used. Accordingly, any scale variability in the growth curves will lead to bias in constant-threshold-based Cqs, making it mandatory that workers should either use scale-insensitive Cqs or normalize their growth curves to constant amplitude before applying the constant threshold method. PMID:27958340

  15. Quantitative Assessment of the CCMC's Experimental Real-time SWMF-Geospace Results

    NASA Astrophysics Data System (ADS)

    Liemohn, Michael; Ganushkina, Natalia; De Zeeuw, Darren; Welling, Daniel; Toth, Gabor; Ilie, Raluca; Gombosi, Tamas; van der Holst, Bart; Kuznetsova, Maria; Maddox, Marlo; Rastaetter, Lutz

    2016-04-01

    Experimental real-time simulations of the Space Weather Modeling Framework (SWMF) are conducted at the Community Coordinated Modeling Center (CCMC), with results available there (http://ccmc.gsfc.nasa.gov/realtime.php), through the CCMC Integrated Space Weather Analysis (iSWA) site (http://iswa.ccmc.gsfc.nasa.gov/IswaSystemWebApp/), and the Michigan SWMF site (http://csem.engin.umich.edu/realtime). Presently, two configurations of the SWMF are running in real time at CCMC, both focusing on the geospace modules, using the BATS-R-US magnetohydrodynamic model, the Ridley Ionosphere Model, and with and without the Rice Convection Model for inner magnetospheric drift physics. While both have been running for several years, nearly continuous results are available since July 2015. Dst from the model output is compared against the Kyoto real-time Dst, in particular the daily minimum value of Dst to quantify the ability of the model to capture storms. Contingency tables are presented, showing that the run with the inner magnetosphere model is much better at reproducing storm-time values. For disturbances with a minimum Dst lower than -50 nT, this version yields a probability of event detection of 0.86 and a Heidke Skill Score of 0.60. In the other version of the SWMF, without the inner magnetospheric module included, the modeled Dst never dropped below -50 nT during the examined epoch.

  16. Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei.

    PubMed

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

    Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: β-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); β-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Evaluation of reference genes for real-time quantitative PCR in the marine flavobacterium Zobellia galactanivorans.

    PubMed

    Thomas, François; Barbeyron, Tristan; Michel, Gurvan

    2011-01-01

    The marine bacteria Zobellia galactanivorans is an emerging model microorganism for the bioconversion of algal polysaccharides. The sequence analysis of its genome opens the way to in-depth gene expression analysis, such as reverse transcription quantitative PCR (RT-qPCR) studies. The selection and validation of reference genes are a mandatory first step for the accurate quantification of transcripts. We selected fourteen candidate reference genes belonging to distinct pathways, namely replication, transcription, translation, citric acid cycle, amino acid, nucleotide and dihydrofolate metabolisms, and peptidoglycan, FMN and aromatic compounds synthesis. We quantified their expression by RT-qPCR in various culture conditions corresponding to different temperatures, carbon sources or stresses. The applications geNorm and Normfinder allowed ranking the genes according to their stability and gave concordant results. We found that the geometric average of the expression of glyA, icdA and gmkA can be confidently used to normalize the transcript abundance of genes of interest. In conclusion, this work provides a reliable procedure for gene expression analysis in the flavobacterium Z. galactanivorans and a validated set of reference genes to be used in future transcriptomics approaches. The strategy developed could also be the starting point for similar studies in other members of the Flavobacteria class.

  18. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

    PubMed Central

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes. PMID:27242878

  19. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

    PubMed

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes.

  20. SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case.

    PubMed

    Frigerio, Marcello; Passeri, Elena; de Filippis, Tiziana; Rusconi, Daniela; Valaperta, Rea; Carminati, Mario; Donnangelo, Anita; Costa, Elena; Persani, Luca; Finelli, Palma; Corbetta, Sabrina

    2011-05-05

    22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the VPREB1 qPCR marker was pointed out. A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH) was used for confirmation. qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The VPREB1 gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the VPREB1 gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the VPREB1 marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion. Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the VPREB1 marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.

  1. SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

    PubMed Central

    2011-01-01

    Background 22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the VPREB1 qPCR marker was pointed out. Methods A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH) was used for confirmation. Results qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The VPREB1 gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the VPREB1 gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the VPREB1 marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion. Conclusions Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the VPREB1 marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations. PMID:21545739

  2. Using a field quantitative real-time PCR test to rapidly identify highly viremic rift valley fever cases.

    PubMed

    Njenga, M Kariuki; Paweska, Janusz; Wanjala, Rose; Rao, Carol Y; Weiner, Matthew; Omballa, Victor; Luman, Elizabeth T; Mutonga, David; Sharif, Shanaaz; Panning, Marcus; Drosten, Christian; Feikin, Daniel R; Breiman, Robert F

    2009-04-01

    Approximately 8% of Rift Valley fever (RVF) cases develop severe disease, leading to hemorrhage, hepatitis, and/or encephalitis and resulting in up to 50% of deaths. A major obstacle in the management of RVF and other viral hemorrhagic fever cases in outbreaks that occur in rural settings is the inability to rapidly identify such cases, with poor prognosis early enough to allow for more-aggressive therapies. During an RVF outbreak in Kenya in 2006 to 2007, we evaluated whether quantitative real-time reverse transcription-PCR (qRT-PCR) could be used in the field to rapidly identify viremic RVF cases with risk of death. In 52 of 430 RVF cases analyzed by qRT-PCR and virus culture, 18 died (case fatality rate [CFR] = 34.6%). Levels of viremia in fatal cases were significantly higher than those in nonfatal cases (mean of 10(5.2) versus 10(2.9) per ml; P < 0.005). A negative correlation between the levels of infectious virus particles and the qRT-PCR crossover threshold (C(T)) values allowed the use of qRT-PCR to assess prognosis. The CFR was 50.0% among cases with C(T) values of <27.0 (corresponding to 2.1 x 10(4) viral RNA particles/ml of serum) and 4.5% among cases with C(T) values of >or=27.0. This cutoff yielded 93.8% sensitivity and a 95.5% negative predictive value; the specificity and positive predictive value were 58% and 50%, respectively. This study shows a correlation between high viremia and fatality and indicates that qRT-PCR testing can identify nearly all fatal RVF cases.

  3. Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

    PubMed Central

    Wang, Jinyao; Kang, Xiuping; Weng, Yiqun

    2017-01-01

    Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the ‘Datong’ landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species. PMID:28362875

  4. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

    PubMed Central

    Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong

    2014-01-01

    The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species. PMID:24823940

  5. Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses

    PubMed Central

    Waggoner, Jesse J.; Abeynayake, Janaki; Sahoo, Malaya K.; Gresh, Lionel; Tellez, Yolanda; Gonzalez, Karla; Ballesteros, Gabriela; Pierro, Anna M.; Gaibani, Paolo; Guo, Frances P.; Sambri, Vittorio; Balmaseda, Angel; Karunaratne, Kumudu; Harris, Eva; Pinsky, Benjamin A.

    2013-01-01

    Background Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. Methodology/Principal Findings An rRT-PCR assay targeting the 5′ untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log10 cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1–4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. Conclusions/Significance This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this

  6. Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

    NASA Technical Reports Server (NTRS)

    Parra, Macarena; Jung, Jimmy; Tran, Luan; Boone, Travis; Almeida, Eduardo; Schonfeld, Julie

    2016-01-01

    The WetLab-2 system was developed by NASA Ames Research Center to offer new capabilities to researchers. The system can lyse cells and extract RNA (Ribonucleic Acid) on-orbit from different sample types ranging from microbial cultures to animal tissues. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (Polymerase Chain Reaction) (qRT-PCR) analysis without the need for sample return. The qRT-PCR results can be downlinked to the ground a few hours after the completion of the run. The validation flight of the WetLab-2 system launched on SpaceX-8 on April 8, 2016. On orbit operations started on April 15th with system setup and was followed by three quantitative PCR runs using an E. coli genomic DNA template pre-loaded at three different concentrations. These runs were designed to discern if quantitative PCR functions correctly in microgravity and if the data is comparable to that from the ground control runs. The flight data showed no significant differences compared to the ground data though there was more variability in the values, this was likely due to the numerous small bubbles observed. The capability of the system to process samples and purify RNA was then validated using frozen samples prepared on the ground. The flight data for both E. coli and mouse liver clearly shows that RNA was successfully purified by our system. The E. coli qRT-PCR run showed successful singleplex, duplex and triplex capability. Data showed high variability in the resulting Cts (Cycle Thresholds [for the PCR]) likely due to bubble formation and insufficient mixing during the procedure run. The mouse liver qRT-PCR run had successful singleplex and duplex reactions and the variability was slightly better as the mixing operation was improved. The ability to purify and stabilize RNA and to conduct qRT-PCR on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. The

  7. Quantitative real-time PCR eliminates false-positives in colony screening PCR.

    PubMed

    Skarratt, Kristen K; Fuller, Stephen J

    2014-01-01

    We report an alternative approach to colony screening using real-time PCR (qPCR) which can be used instead of the traditional end-point PCR to eliminate false-positives and reduce processing times. False-positive transformants can easily be distinguished from true-positives by comparing Ct values derived from qPCR amplification curves. In addition, the use of qPCR allows for more efficient processing since a gel electrophoresis step is not required and the screening process is no longer limited by the capacity of the gel apparatus.

  8. Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

    PubMed

    Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

    2009-01-01

    The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

  9. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

    PubMed Central

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

  10. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species.

    PubMed

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-08-26

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management.

  11. Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

    PubMed

    Fekri Soofi Abadi, Maryam; Dabiri, Shahriar; Fotouhi Ardakani, Reza; Fani Malaki, Lina; Amirpoor Rostami, Sahar; Ziasistani, Mahsa; Dabiri, Donya

    2016-07-01

    Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

  12. Effect of ionizing radiation on the quantitative detection of Salmonella using real-time PCR

    NASA Astrophysics Data System (ADS)

    Lim, Sangyong; Jung, Jinwoo; Kim, Minjeong; Ryu, Sangryeol; Kim, Dongho

    2008-09-01

    Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold ( CT) increased 1-1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared CT values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3-3.3-fold underestimation of bacterial counts with respect to irradiation dose.

  13. Real-time probe based quantitative determination of material properties at the nanoscale.

    PubMed

    Saraswat, G; Agarwal, P; Haugstad, G; Salapaka, M V

    2013-07-05

    Tailoring the properties of a material at the nanoscale holds the promise of achieving hitherto unparalleled specificity of the desired behavior of the material. Key to realizing this potential of tailoring materials at the nanoscale are methods for rapidly estimating physical properties of the material at the nanoscale. In this paper, we report a method for simultaneously determining the topography, stiffness and dissipative properties of materials at the nanoscale in a probe based dynamic mode operation. The method is particularly suited for investigating soft-matter such as polymers and bio-matter. We use perturbation analysis tools for mapping dissipative and stiffness properties of material into parameters of an equivalent linear time-invariant model. Parameters of the equivalent model are adaptively estimated, where, for robust estimation, a multi-frequency excitation of the probe is introduced. We demonstrate that the reported method of simultaneously determining multiple material properties can be implemented in real-time on existing probe based instruments. We further demonstrate the effectiveness of the method by investigating properties of a polymer blend in real-time.

  14. Development of a Real-Time Quantitative PCR for Detecting Duck Hepatitis A Virus Genotype C

    PubMed Central

    Huang, Qiuxue; Yue, Hua; Zhang, Bin; Nie, Peiting

    2012-01-01

    Recently, duck hepatitis A virus genotype C (DHAV-C), a causative agent of duck viral hepatitis, has been responsible for increasing economic losses in the duck industry in China and South Korea. In this study, a real-time PCR assay targeting the 2C gene for detecting DHAV-C was developed. The assay was confirmed to be specific and sensitive, and the minimum detection limit was 3.36 × 103 copies per reaction, making this assay suitable for rapid diagnosis of DHAV-C infection from clinical samples. In addition, the dynamics of the viral loads in tissues of specific-pathogen-free (SPF) ducklings infected with DHAV-C were investigated using this method. The DHAV-C could be detected earliest in the liver within 12 h postinfection. Moreover, high viral loads were identified in the heart, liver, spleen, lung, kidney, bursa of Fabricius, thymus, pancreas, brain, and small intestine after 24 h postinfection. Taking the data collectively, the study described in this report is the first to have developed a real-time PCR method for detection of DHAV-C and thus contributes to pathogenicity research. PMID:22855514

  15. Development of a real-time quantitative PCR for detecting duck hepatitis a virus genotype C.

    PubMed

    Huang, Qiuxue; Yue, Hua; Zhang, Bin; Nie, Peiting; Tang, Cheng

    2012-10-01

    Recently, duck hepatitis A virus genotype C (DHAV-C), a causative agent of duck viral hepatitis, has been responsible for increasing economic losses in the duck industry in China and South Korea. In this study, a real-time PCR assay targeting the 2C gene for detecting DHAV-C was developed. The assay was confirmed to be specific and sensitive, and the minimum detection limit was 3.36 × 10(3) copies per reaction, making this assay suitable for rapid diagnosis of DHAV-C infection from clinical samples. In addition, the dynamics of the viral loads in tissues of specific-pathogen-free (SPF) ducklings infected with DHAV-C were investigated using this method. The DHAV-C could be detected earliest in the liver within 12 h postinfection. Moreover, high viral loads were identified in the heart, liver, spleen, lung, kidney, bursa of Fabricius, thymus, pancreas, brain, and small intestine after 24 h postinfection. Taking the data collectively, the study described in this report is the first to have developed a real-time PCR method for detection of DHAV-C and thus contributes to pathogenicity research.

  16. Real time quantitative colourimetric test for methamphetamine detection using digital and mobile phone technology.

    PubMed

    Choodum, Aree; Parabun, Kaewalee; Klawach, Nantikan; Daeid, Niamh Nic; Kanatharana, Proespichaya; Wongniramaikul, Worawit

    2014-02-01

    The Simon presumptive color test was used in combination with the built-in digital camera on a mobile phone to detect methamphetamine. The real-time Red-Green-Blue (RGB) basic color data was obtained using an application installed on the mobile phone and the relationship profile between RGB intensity, including other calculated values, and the colourimetric product was investigated. A wide linear range (0.1-2.5mg mL(-1)) and a low detection limit (0.0110±0.0001-0.044±0.002mg mL(-1)) were achieved. The method also required a small sample size (20μL). The results obtained from the analysis of illicit methamphetamine tablets were comparable to values obtained from gas chromatograph-flame ionization detector (GC-FID) analysis. Method validation indicated good intra- and inter-day precision (2.27-4.49%RSD and 2.65-5.62%RSD, respectively). The results suggest that this is a powerful real-time mobile method with the potential to be applied in field tests. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. Analysis of THCA synthase gene expression in cannabis: a preliminary study by real-time quantitative PCR.

    PubMed

    Cascini, Fidelia; Passerotti, Stella; Boschi, Ilaria

    2013-09-10

    In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression. Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability. As results we found minor gene expression in samples from leaves of young plants. Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants.

  18. The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE).

    PubMed

    Taylor, Sean C; Mrkusich, Eli M

    2014-01-01

    In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR.

  19. Real time measurements of elongation by a reverse transcriptase using surface plasmon resonance.

    PubMed Central

    Buckle, M; Williams, R M; Negroni, M; Buc, H

    1996-01-01

    A rapid direct assay for polymerase-induced elongation along a given template is an obligate requirement for understanding the processivity of polymerization and the mode of action of drugs and inhibitors on this process. Surface plasmon resonance can be used to follow the association and the dissociation rates of a given reverse transcriptase on DNA.RNA and DNA.DNA hybrids immobilized on a biotin-streptavidin surface. The addition of nucleotides complementary to the template strand produces an increase in the local mass, as deduced from an increase in the measured signal, due to elongation of the primer strand that allows an estimation of both the extent and rate of the polymerization process. The terminator drug 3'-deoxy-3'-azidothymidine triphosphate completely abolishes the increase in signal as would be expected from an inhibition of elongation. This technique provides a sensitive assay for the affinities of different polymerases for specific templates and for the effects of terminators of the elongation process. PMID:8570654

  20. [HTLV-1 and HTLV-2 proviral load: a simple method using quantitative real-time PCR].

    PubMed

    Tamegão-Lopes, Bruna Pedroso; Rezende, Priscila Rocha; Maradei-Pereira, Luciana Maria Cunha; de Lemos, José Alexandre Rodrigues

    2006-01-01

    When the human T cell lymphotropic virus (HTLV) is integrated with the host cell genome (provirus), its proviral DNA is a replication marker. Proviral load appears to be an important factor in the development of diseases related to these retroviruses. In this study, a methodology for absolute quantification of the HTLV-1 and HTLV-2 proviral load using real-time PCR was developed. Fifty-three blood donor samples with positive ELISA test result were subjected to this methodology, which utilized the TaqMan system for three target sequences: HTLV-1, HTLV-2 and albumin. The absolute proviral load was quantified using the relative ratio between the HTLV genome and the host cell genome, taking into consideration the white blood cell count. The method presented is sensitive (215 copies/ml), practical and simple for proviral quantification, and is efficient and appropriate for confirming and discriminating infections according to viral type.

  1. Quantitative 'real-time' imaging of multi-phase flow in ceramic monoliths.

    PubMed

    Sederman, A J; Mantle, M D; Gladden, L F

    2003-01-01

    An extension of the RARE technique has been developed which acquires multiple images from a single radio-frequency excitation. This pulse sequence has been used to image, in real-time, gas flow through stagnant liquid within parallel-channel ceramic monoliths. From these images, gas-phase volume fractions, and distributions of gas bubble length and velocity as a function of gas flow rate (50-300 cm3 min(-1)) and channel size (300 and 400 channels per square inch, cpsi) are obtained directly. Increasing the gas flow rate increased the number of large bubbles and the average bubble velocity. A bimodal distribution in the bubble velocities was observed for flow within the larger channel size (300 cpsi) in contrast to a broad unimodal distribution characterizing two-phase flow within the smaller channel size (400 cpsi).

  2. Sex ratio determination in bovine semen: a new approach by quantitative real time PCR.

    PubMed

    Parati, K; Bongioni, G; Aleandri, R; Galli, A

    2006-12-01

    Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction. Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies. In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV=2.58%) and reproducibility (CV=2.57%) was shown. The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P>0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11+/-0.56% for ejaculates and 50.17+/-0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.

  3. Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus.

    PubMed

    Buitrago, Dolores; Rocha, Ana; Tena-Tomás, Cristina; Vigo, Marta; Agüero, Montserrat; Jiménez-Clavero, Miguel Angel

    2012-09-01

    In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5'-Taq nuclease-3' minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

  4. Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens▿

    PubMed Central

    Boddicker, Jennifer D.; Rota, Paul A.; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B.; Thompson, Robert; Bellini, William J.; Pentella, Michael; DesJardin, Lucy E.

    2007-01-01

    The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques. PMID:17652480

  5. Quantitative detection of Cucumber vein yellowing virus in susceptible and partially resistant plants using real-time PCR.

    PubMed

    Picó, Belén; Sifres, Alicia; Nuez, Fernando

    2005-09-01

    A method for the detection of Cucumber vein yellowing virus (CVYV) that combines reverse transcription with real-time PCR (SYBR((R)) Green chemistry) was developed using specific primers designed from a nucleotide sequence of the RNA polymerase gene (NIb) conserved among all the available CVYV strains. This method provided a linear assay over five to six orders of magnitude and reproducibly detected titres as low as 10(3) molecules of the target CVYV cDNA. Real-time PCR gave reproducible results for the quantification of CVYV in young leaves of susceptible and resistant cucumber landraces after mechanical inoculation. Significant differences in the starting amount of target cDNA were found between the analyzed genotypes, indicating differences in viral accumulation that correlated to their different levels of resistance. Real-time PCR results validated our previous findings using slot-blot hybridization, the dominance of the strong resistance to CVYV displayed by C.sat 10, and provided improved reliability and sensitivity of detection. This method has great potential in resistance breeding for germplasm screening, characterization of resistance mechanisms and genetic studies.

  6. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    USGS Publications Warehouse

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W.; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  7. Quantitative real-time analysis of collective cancer invasion and dissemination

    NASA Astrophysics Data System (ADS)

    Ewald, Andrew J.

    2015-05-01

    A grand challenge in biology is to understand the cellular and molecular basis of tissue and organ level function in mammals. The ultimate goals of such efforts are to explain how organs arise in development from the coordinated actions of their constituent cells and to determine how molecularly regulated changes in cell behavior alter the structure and function of organs during disease processes. Two major barriers stand in the way of achieving these goals: the relative inaccessibility of cellular processes in mammals and the daunting complexity of the signaling environment inside an intact organ in vivo. To overcome these barriers, we have developed a suite of tissue isolation, three dimensional (3D) culture, genetic manipulation, nanobiomaterials, imaging, and molecular analysis techniques to enable the real-time study of cell biology within intact tissues in physiologically relevant 3D environments. This manuscript introduces the rationale for 3D culture, reviews challenges to optical imaging in these cultures, and identifies current limitations in the analysis of complex experimental designs that could be overcome with improved imaging, imaging analysis, and automated classification of the results of experimental interventions.

  8. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR.

    PubMed

    Bushon, R N; Kephart, C M; Koltun, G F; Francy, D S; Schaefer, F W; Alan Lindquist, H D

    2010-03-01

    The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.

  9. Quantitative Analysis Of Sperm Motion Kinematics From Real-Time Video-Edge Images

    NASA Astrophysics Data System (ADS)

    Davis, Russell O...; Katz, David F.

    1988-02-01

    A new model of sperm swimming kinematics, which uses signal processing methods and multivariate statistical techniques to identify individual cell-motion parameters and unique cell populations, is presented. Swimming paths of individual cells are obtained using real-time, video-edge digitization. Raw paths are adaptively filtered to identify average paths, and measurements of space-time oscillations about average paths are made. Time-dependent frequency information is extracted from spatial variations about average paths using harmonic analysis. Raw-path and average-path measures such as curvature, curve length, and straight-line length, and measures of oscillations about average paths such as time-dependent amplitude and frequency variations, are used in a multivariate, cluster analysis to identify unique cell populations. The entire process, including digitization of sperm video images, is computer-automated. Preliminary results indicate that this method of tracking, digitization, and kinematic analysis accurately identifies unique cell subpopulations, including: the relative numbers of cells in each subpopulation, how subpopulations differ, and the extent and significance of such differences. With appropriate work, this approach may be useful for clinical discrimination between normal and abnormal semen specimens.

  10. A model for real-time quantitative rainfall forecasting using remote sensing. 1. Formulation

    NASA Astrophysics Data System (ADS)

    French, Mark N.; Krajewski, Witold F.

    1994-04-01

    A physically based rainfall forecasting model for real-time hydrologic applications is developed with emphasis on utilization of remote sensing observations. Temporal and spatial scales of interest are lead times of the order of hours and areas of the order of 10 km2. The dynamic model is derived from conservation of mass in a cloud column as defined by the continuity equations for air, liquid water, water vapor, and cloud water. Conservation of momentum is modeled using a semi-Lagrangian frame of reference. The model state is vertically integrated liquid water content in a column of the atmosphere. Additionally, laws of thermodynamics, adiabatic air parcel theory, and cloud microphysics are applied to derive a basic parameterization of the governing equations of model dynamics. The parameterization is in terms of hydrometeorologic observables including radar reflectivity, satellite-infrared brightness temperature, and ground-level air temperature, dew point temperature, and pressure. Implementation and application is described by French et al. (this issue) and involves incorporation of uncertainty analysis and a two-dimensional spatial domain, where the dynamics of the continuous space-time rainfall process are discretized onto a rectangular grid.

  11. Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction, Sierra Leone, 2014.

    PubMed

    Liu, Licheng; Sun, Yang; Kargbo, Brima; Zhang, Chuntao; Feng, Huahua; Lu, Huijun; Liu, Wenseng; Wang, Chengyu; Hu, Yi; Deng, Yongqiang; Jiang, Jiafu; Kang, Xiaoping; Yang, Honglei; Jiang, Yongqiang; Yang, Yinhui; Kargbo, David; Qian, Jun; Chen, Weijun

    2015-09-15

    During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus.

  12. Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

    PubMed

    Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier

    2015-07-01

    Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium.

  13. Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

    PubMed

    Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

    2011-09-01

    To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

  14. Automated extraction and quantitation of oncogenic HPV genotypes from cervical samples by a real-time PCR-based system.

    PubMed

    Broccolo, Francesco; Cocuzza, Clementina E

    2008-03-01

    Accurate laboratory assays for the diagnosis of persistent oncogenic HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. HPV viral load has been suggested to be a surrogate marker of persistent infection. Four independent real-time quantitative TaqMan PCR assays were developed for: HPV-16, -31, -18 and/or -45 and -33 and/or -52, -58, -67. The assays had a wide dynamic range of detection and a high degree of accuracy, repeatability and reproducibility. In order to minimize material and hands-on time, automated nucleic acid extraction was performed using a 96-well plate format integrated into a robotic liquid handler workstation. The performance of the TaqMan assays for HPV identification was assessed by comparing results with those obtained by means of PCR using consensus primers (GP5+/GP6+) and sequencing (296 samples) and INNO-LiPA analysis (31 samples). Good agreement was found generally between results obtained by real-time PCR assays and GP(+)-PCR system (kappa statistic=0.91). In conclusion, this study describes four newly developed real-time PCR assays that provide a reliable and high-throughput method for detection of not only HPV DNA but also HPV activity of the most common oncogenic HPV types in cervical specimens.

  15. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

    PubMed

    Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

    2010-03-01

    Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

  16. Development and application of a quantitative real-time PCR assay to detect feline leukemia virus RNA.

    PubMed

    Torres, Andrea N; O'Halloran, Kevin P; Larson, Laurie J; Schultz, Ronald D; Hoover, Edward A

    2008-05-15

    We previously defined four categories of feline leukemia virus (FeLV) infection, designated as abortive, regressive, latent, and progressive. To determine if detectable viral DNA is transcriptionally active in the absence of antigenemia, we developed and validated a real-time viral RNA qPCR assay. This assay proved to be highly sensitive, specific, reproducible, and allowed reliable quantitation. We then applied this methodology, together with real-time DNA qPCR and p27 capsid antigen capture ELISA, to examine cats challenged with FeLV. We found that circulating viral RNA and DNA levels were highly correlated and the assays were almost in perfect agreement. This indicates that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia. The real-time qPCR assays are more sensitive than the most commonly used FeLV diagnostic assay, the p27 capsid antigen capture ELISA. Application of qPCR assays may add greater depth in understanding of FeLV-host relationships.

  17. Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis.

    PubMed

    Gulden, Robert H; Lerat, Sylvain; Hart, Miranda M; Powell, Jeff R; Trevors, Jack T; Pauls, K Peter; Klironomos, John N; Swanton, Clarence J

    2005-07-27

    Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 degrees C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 degrees C).

  18. Real-time RT-PCR for the detection and quantitative analysis of equine rhinitis viruses.

    PubMed

    Quinlivan, M; Maxwell, G; Lyons, P; Arkins, S; Cullinane, A

    2010-03-01

    Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. To develop a sensitive, rapid, real-time RT-PCR (rRT-PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5' UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT-PCR were verified by virus isolation and ERBV positive samples were verified by rRT-PCR using a different set of primers. The detection limit of the rRT-PCR for both viruses was 10-100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co-circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT-PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp). The rRT-PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV. These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.

  19. Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA content in food.

    PubMed

    Brodmann, Peter D; Ilg, Evelyn C; Berthoud, Hélène; Herrmann, Andre

    2002-01-01

    Quantitative detection methods are needed for enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients. This labeling threshold, which is set to 1% in the European Union and Switzerland, must be applied to all approved GMOs. Four different varieties of maize are approved in the European Union: the insect-resistant Bt176 maize (Maximizer), Btl 1 maize, Mon810 (YieldGard) maize, and the herbicide-tolerant T25 (Liberty Link) maize. Because the labeling must be considered individually for each ingredient, a quantitation system for the endogenous maize content is needed in addition to the GMO-specific detection systems. Quantitative real-time polymerase chain reaction detection methods were developed for the 4 approved genetically modified maize varieties and for an endogenous maize (invertase) gene system.

  20. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    PubMed

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

  1. Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction.

    PubMed

    Rodríguez, Miguel A; García, Teresa; González, Isabel; Asensio, Luis; Hernández, Pablo E; Martín, Rosario

    2004-03-24

    A real-time quantitative Polymerase Chain Reaction (PCR) method has been developed for the quantitation of mule duck (Anas platyrhynchos x Cairina moschata) in binary duck/goose foie gras mixtures. The method combines the use of real-time PCR with duck-specific and endogenous control "duck + goose" primers to measure duck content and total foie gras content, respectively. Both PCR systems (duck-specific and duck + goose) were designed on the mitochondrial 12S ribosomal RNA gene (rRNA). The duck-specific system amplifies a 96 bp fragment from duck DNA, whereas the duck + goose system amplifies a 120 bp fragment from duck and goose DNA. The method measures PCR product accumulation through a FAM-labeled fluorogenic probe (TaqMan). The C(t) (threshold cycle) values obtained from the duck + goose system are used to normalize the ones obtained from the duck-specific system. Analysis of experimental duck/goose foie gras binary mixtures demonstrated the suitability of the assay for the detection and quantitation of duck in the range of 1-25%. This genetic marker can be very useful to avoid mislabeling or fraudulent species substitution of goose by duck in foie gras.

  2. Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

    NASA Astrophysics Data System (ADS)

    Walsh, Adrian A.

    2017-04-01

    Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

  3. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions.

    PubMed

    Zhu, Jianfeng; Zhang, Lifeng; Li, Wanfeng; Han, Suying; Yang, Wenhua; Qi, Liwang

    2013-01-01

    Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.

  4. Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR.

    PubMed

    Oliveira, Sara R; Vieira, Helena L A; Duarte, Carlos B

    2015-09-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results

  5. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    PubMed Central

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

  6. Endonuclease Restriction-Mediated Real-Time Polymerase Chain Reaction: A Novel Technique for Rapid, Sensitive and Quantitative Detection of Nucleic-Acid Sequence

    PubMed Central

    Wang, Yi; Wang, Yan; Zhang, Lu; Li, Machao; Luo, Lijuan; Liu, Dongxin; Li, Hua; Cao, Xiaolong; Hu, Shoukui; Jin, Dong; Xu, Jianguo; Ye, Changyun

    2016-01-01

    The article reported a novel methodology for real-time PCR analysis of nucleic acids, termed endonuclease restriction-mediated real-time polymerase chain reaction (ET-PCR). Just like PCR, ET-PCR only required one pair of primers. A short sequence, which was recognized by restriction enzyme BstUI, was attached to the 5′ end of the forward (F) or reverse (R) PCR primer, and the new F or R primer was named EF or ER. EF/ER was labeled at the 5′ end with a reporter dye and in the middle with a quenching dye. BstUI cleaves the newly synthesized double-stranded terminal sequences (5′ end recognition sequences and their complementary sequences) during the extension phase, which separates the reporter molecule from the quenching dye, leading to a gain of fluorescence signal. This process is repeated in each amplification cycle and unaffected the exponential synthesis of the PCR amplification. ET-PCR allowed real-time analysis of single or multiple targets in a single vessel, and provided the reproducible quantitation of nucleic acids. The analytical sensitivity and specificity of ET-PCR were successfully evaluated, detecting down to 250 fg of genomic DNA per tube of target pathogen DNA examined, and the positive results were generated in a relatively short period. Moreover, the practical application of ET-PCR for simultaneous detection of multiple target pathogens was also demonstrated in artificially contaminated blood samples. In conclusion, due to the technique’s simplicity of design, reproducible data and low contamination risk, ET-PCR assay is an appealing alternative to conventional approaches currently used for real-time nucleic acid analysis. PMID:27468284

  7. Endonuclease Restriction-Mediated Real-Time Polymerase Chain Reaction: A Novel Technique for Rapid, Sensitive and Quantitative Detection of Nucleic-Acid Sequence.

    PubMed

    Wang, Yi; Wang, Yan; Zhang, Lu; Li, Machao; Luo, Lijuan; Liu, Dongxin; Li, Hua; Cao, Xiaolong; Hu, Shoukui; Jin, Dong; Xu, Jianguo; Ye, Changyun

    2016-01-01

    The article reported a novel methodology for real-time PCR analysis of nucleic acids, termed endonuclease restriction-mediated real-time polymerase chain reaction (ET-PCR). Just like PCR, ET-PCR only required one pair of primers. A short sequence, which was recognized by restriction enzyme BstUI, was attached to the 5' end of the forward (F) or reverse (R) PCR primer, and the new F or R primer was named EF or ER. EF/ER was labeled at the 5' end with a reporter dye and in the middle with a quenching dye. BstUI cleaves the newly synthesized double-stranded terminal sequences (5' end recognition sequences and their complementary sequences) during the extension phase, which separates the reporter molecule from the quenching dye, leading to a gain of fluorescence signal. This process is repeated in each amplification cycle and unaffected the exponential synthesis of the PCR amplification. ET-PCR allowed real-time analysis of single or multiple targets in a single vessel, and provided the reproducible quantitation of nucleic acids. The analytical sensitivity and specificity of ET-PCR were successfully evaluated, detecting down to 250 fg of genomic DNA per tube of target pathogen DNA examined, and the positive results were generated in a relatively short period. Moreover, the practical application of ET-PCR for simultaneous detection of multiple target pathogens was also demonstrated in artificially contaminated blood samples. In conclusion, due to the technique's simplicity of design, reproducible data and low contamination risk, ET-PCR assay is an appealing alternative to conventional approaches currently used for real-time nucleic acid analysis.

  8. Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection

    PubMed Central

    Gruber, Franz; Falkner, Falko G.; Dorner, Friedrich; Hämmerle, Thomas

    2001-01-01

    A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 102 to at least 5 × 106 copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets. PMID:11375203

  9. [Use of real-time PCR for quantitative assessment of lactic acid bacteria and bifidobacteria in dairy products].

    PubMed

    Zelenaia, L B; Kovalenko, N K; Oblap, R V; Hovak, N B; Golubets, R A

    2012-01-01

    Composition of lactic acid bacteria and bifidobacteria in raw milk and home-made milk products has been analyzed using real-time PCR (quantitative PCR) with genus-specific primers to Enterococcus, Lactobacillus and Bifidobacteria. Bacteria belonging to these genera have been revealed in all samples analyzed (milk, sour cream, cottage cheese). It has been shown that the representatives of Enterococcus and Lactobacillus genera dominated in the samples analyzed (10(3)-10(7) genome equivalent/ml (mg)). The largest number of these microorganisms (10(7) genome equivalent/mg) has been detected in cottage cheese.

  10. Comparison of two real-time quantitative polymerase chain reaction strategies for minimal residual disease evaluation in lymphoproliferative disorders: correlation between immunoglobulin gene mutation load and real-time quantitative polymerase chain reaction performance.

    PubMed

    Della Starza, Irene; Cavalli, Marzia; Del Giudice, Ilaria; Barbero, Daniela; Mantoan, Barbara; Genuardi, Elisa; Urbano, Marina; Mannu, Claudia; Gazzola, Anna; Ciabatti, Elena; Guarini, Anna; Foà, Robin; Galimberti, Sara; Piccaluga, Pierpaolo; Gaidano, Gianluca; Ladetto, Marco; Monitillo, Luigia

    2014-09-01

    We compared two strategies for minimal residual disease evaluation of B-cell lymphoproliferative disorders characterized by a variable immunoglobulin heavy chain (IGH) genes mutation load. Twenty-five samples from chronic lymphocytic leukaemia (n = 18) or mantle cell lymphoma (n = 7) patients were analyzed. Based on IGH variable region genes, 22/25 samples carried > 2% mutations, 20/25 > 5%. In the IGH joining region genes, 23/25 samples carried > 2% mutations, 18/25 > 5%. Real-time quantitative polymerase chain reaction was performed on IGH genes using two strategies: method A utilizes two patient-specific primers, whereas method B employs one patient-specific and one germline primer, with different positions on the variable, diversity and joining regions. Twenty-three samples (92%) resulted evaluable using method A, only six (24%) by method B. Method B poor performance was specifically evident among mutated IGH variable/joining region cases, although no specific mutation load above, which the real-time quantitative polymerase chain reaction failed was found. The molecular strategies for minimal residual disease evaluation should be adapted to the B-cell receptor features of the disease investigated.

  11. Rapid Detection of Enterovirus RNA in Cerebrospinal Fluid Specimens with a Novel Single-Tube Real-Time Reverse Transcription-PCR Assay

    PubMed Central

    Verstrepen, Walter A.; Kuhn, Sofie; Kockx, Mark M.; Van De Vyvere, Martine E.; Mertens, An H.

    2001-01-01

    A single-tube real-time reverse transcription-PCR (RT-PCR) assay for enterovirus detection in cerebrospinal fluid (CSF) was developed based on a fluorogenic probe and primers directed to highly conserved sequences in the 5′ untranslated region of the enterovirus genome. Quantitative detection of enterovirus genome was demonstrated in a linear range spanning at least 5 logs. Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) corresponding in our current extraction protocol to 592 enterovirus genome equivalents per ml of CSF. Twenty CSF specimens not suspected of viral meningitis were all found to be negative, and no cross-reactivity with herpes simplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PCR (27.1%), whereas only 17 were found to be positive by viral culture (24.3%). The sensitivity of the assay was 100% and the specificity was 96.2% compared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may offer a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct required considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovirus detection assays. PMID:11682535

  12. SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase.

    PubMed

    Kokkula, Chakradhar; Palanisamy, Navaneethan; Ericstam, Malin; Lennerstrand, Johan

    2016-10-01

    There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.

  13. Verus: A Tool for Quantitative Analysis of Finite-State Real-Time Systems.

    DTIC Science & Technology

    1996-08-12

    and A. P. Sistla . Automatic verification of finite-state concurrent systems using temporal logic specifications. ACM TOPLAS, 8(2):244–263, 1986. [6] E...Proceedings of the 11th CHDL, 1993. [7] E. A. Emerson, A. K. Mok, A. P. Sistla , and J. Srinivasan. Quantitative temporal reasoning. In Lecture Notes in

  14. Computing Quantitative Characteristics of Finite-State Real-Time Systems

    DTIC Science & Technology

    1994-05-04

    Emerson, and A. P. Sistla . Automatic verification of finite-state concurrent sys- tems using temporal logic specifications. ACM Transactions on...Programming Languages and Systems, 8(2):244-263, 1986. [8] E. A. Emerson, A. K. Mok, A. P. Sistla , and J. Srinivasan. Quantitative temporal reasoning. In

  15. A Quantitative Approach to the Formal Verification of Real-Time Systems.

    DTIC Science & Technology

    1996-09-01

    my parents Lia and Daniel and to my sister Daniela , for the help and support through- out my whole life. Even though they have not been present during...transient overload, scheduling of aperiodic tasks and priority granularity in communication scheduling [49]. For this rea - son, static scheduling algorithms...Quantitative temporal rea - soning. In Lecture Notes in Computer Science, Computer-Aided Verification. Springer- Verlag, 1990. [34] J. Fernandez, H

  16. Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

    PubMed Central

    Wong, Wilson; Farr, Ryan; Joglekar, Mugdha; Januszewski, Andrzej; Hardikar, Anandwardhan

    2015-01-01

    Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput. PMID:25938938

  17. Comparative evaluation of three commercial quantitative cytomegalovirus standards by use of digital and real-time PCR.

    PubMed

    Hayden, R T; Gu, Z; Sam, S S; Sun, Y; Tang, L; Pounds, S; Caliendo, A M

    2015-05-01

    The recent development of the 1st WHO International Standard for human cytomegalovirus (CMV) and the introduction of commercially produced secondary standards have raised hopes of improved agreement among laboratories performing quantitative PCR for CMV. However, data to evaluate the trueness and uniformity of secondary standards and the consistency of results achieved when these materials are run on various assays are lacking. Three concentrations of each of the three commercially prepared secondary CMV standards were tested in quadruplicate by three real-time and two digital PCR methods. The mean results were compared in a pairwise fashion with nominal values provided by each manufacturer. The agreement of results among all methods for each sample and for like concentrations of each standard was also assessed. The relationship between the nominal values of standards and the measured values varied, depending upon the assay used and the manufacturer of the standards, with the degree of bias ranging from +0.6 to -1.0 log10 IU/ml. The mean digital PCR result differed significantly among the secondary standards, as did the results of the real-time PCRs, particularly when plotted against nominal log10 IU values. Commercially available quantitative secondary CMV standards produce variable results when tested by different real-time and digital PCR assays, with various magnitudes of bias compared to nominal values. These findings suggest that the use of such materials may not achieve the intended uniformity among laboratories measuring CMV viral load, as envisioned by adaptation of the WHO standard. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Comparative Evaluation of Three Commercial Quantitative Cytomegalovirus Standards by Use of Digital and Real-Time PCR

    PubMed Central

    Gu, Z.; Sam, S. S.; Sun, Y.; Tang, L.; Pounds, S.; Caliendo, A. M.

    2015-01-01

    The recent development of the 1st WHO International Standard for human cytomegalovirus (CMV) and the introduction of commercially produced secondary standards have raised hopes of improved agreement among laboratories performing quantitative PCR for CMV. However, data to evaluate the trueness and uniformity of secondary standards and the consistency of results achieved when these materials are run on various assays are lacking. Three concentrations of each of the three commercially prepared secondary CMV standards were tested in quadruplicate by three real-time and two digital PCR methods. The mean results were compared in a pairwise fashion with nominal values provided by each manufacturer. The agreement of results among all methods for each sample and for like concentrations of each standard was also assessed. The relationship between the nominal values of standards and the measured values varied, depending upon the assay used and the manufacturer of the standards, with the degree of bias ranging from +0.6 to −1.0 log10 IU/ml. The mean digital PCR result differed significantly among the secondary standards, as did the results of the real-time PCRs, particularly when plotted against nominal log10 IU values. Commercially available quantitative secondary CMV standards produce variable results when tested by different real-time and digital PCR assays, with various magnitudes of bias compared to nominal values. These findings suggest that the use of such materials may not achieve the intended uniformity among laboratories measuring CMV viral load, as envisioned by adaptation of the WHO standard. PMID:25694529

  19. Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR

    USGS Publications Warehouse

    Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.

    2002-01-01

    A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.

  20. Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy.

    PubMed

    Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Brusin, Martina Rosa; Finotti, Serena; Paderi, Giulia; Gabiano, Clara

    2017-04-01

    Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

  1. DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR.

    PubMed

    Köchl, Silvano; Niederstätter, Harald; Parson, Walther

    2005-01-01

    Forensic laboratories are increasingly confronted with problematic samples from the scene of crime, containing only minute amounts of deoxyribonucleic acid (DNA), which may include polymerase chain reaction (PCR)-inhibiting substances. Efficient DNA extraction procedures, as well as accurate DNA quantification methods, are critical steps involved in the process of successful DNA analysis of such samples. The phenol-chloroform method is a sensitive method for the extraction of DNA from a wide variety of forensic samples, although it is known to be laborious compared with single-tube extraction methods. The relatively high DNA recovery and the quality of the extracted DNA speak for itself. For reliable and sensitive DNA quantitation, the application of real-time PCR is described. We modified a published real-time PCR assay, which allows for the combined analysis of nuclear and mitochondrial DNA, by introducing 1) improved hybridization probes with the use of minor groove binders; 2) an internal positive control (for both nuclear and mitochondrial DNA) for the detection of PCR inhibitors; and 3) different amplicon lengths for the determination of the degradation state of the DNA. The internal positive controls were constructed by site directed mutagenesis by overlap extension of the wild-type mitochondrial and nuclear DNA target with the advantage that no additional probes, which are cost-intensive, are required. The quantitation system is accomplished as a modular concept, which allows for the combined determination of the above-mentioned features (quantity/inhibition or quantity/degradation) depending on the situation.

  2. Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    PubMed

    Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2011-01-01

    To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

  3. Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system.

    PubMed

    Volle, Romain; Nourrisson, Céline; Mirand, Audrey; Regagnon, Christel; Chambon, Martine; Henquell, Cécile; Bailly, Jean-Luc; Peigue-Lafeuille, Hélène; Archimbaud, Christine

    2012-10-01

    Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections.

  4. TaqMan real-time quantitative PCR assay for detection of fluoroquinolone-resistant Neisseria gonorrhoeae.

    PubMed

    Zhao, LiHong; Zhao, ShuPing

    2012-12-01

    It is noted that more than 99 % of fluoroquinolone resistance in Neisseria gonorrhoeae (QRNG) specimens have been shown to have the mutation of Ser91/Phe in the gyrA gene. In order to detect QRNG isolates as quickly as possible, the real-time TaqMan quantitative PCR assay was established for detection of the point mutation of Ser91/Phe in gyrA gene. The standard curve was generated automatically on ABI Prism PE7500. The correlation coefficient (r) of the standard curve was -0.9984 (R(2) = 0.9968), indicating a quietly precise log-linear relationship between the concentration of target DNA and the Ct value. Presently, correlated, cultured antimicrobial susceptibility testing of N. gonorrhoeae isolates continues to be the gold standard method for the detection of antimicrobial resistance. Comparison to the correlated, cultured antimicrobial susceptibility testing, the sensitivity and specificity of the established TaqMan assay for the detection of the QRNG specimens were 100 and 99 %, respectively. The TaqMan assay also allows for rapid detection of QRNG isolates without complex laboratory techniques. Therefore, real-time TaqMan quantitative PCR assay is a rapid, simple, highly sensitive, highly specific, and easy-to-perform method for the detection of the QRNG specimens. It can be applied as a quick screening method for QRNG isolates to help clinical determination of optimal treatment prescription.

  5. A Quantitative Real-Time PCR-Based Strategy for Molecular Evaluation of Nicotine Conversion in Burley Tobacco

    PubMed Central

    Sun, Bo; Xue, Sheng-Ling; Zhang, Fen; Luo, Zhao-Peng; Wu, Ming-Zhu; Chen, Qing; Tang, Hao-Ru; Lin, Fu-Cheng; Yang, Jun

    2015-01-01

    Nornicotine production in Nicotiana tabacum is undesirable because it is the precursor of the carcinogen N′-nitrosonornicotine. In some individual burley tobacco plants, a large proportion of the nicotine can be converted to nornicotine, and this process of nicotine conversion is mediated primarily by enzymatic N-demethylation of nicotine which is controlled mainly by CYP82E4. Here we report a novel strategy based on quantitative real-time polymerase chain reaction (qPCR) method, which analyzed the ratio of nicotine conversion through examining the transcript level of CYP82E4 in burley leaves and do not need ethylene induction before detected. The assay was linear in a range from 1 × 101 to 1 × 105 copies/mL of serially diluted standards, and also showed high specificity and reproducibility (93%–99%). To assess its applicability, 55 plants of burley cultivar Ky8959 at leaf maturing stage were analyzed, and the results were in accordance with those from gas chromatograph-mass spectrometry (GC-MS) method. Moreover, a linear correlation existed between conversion level and CYP82E4 transcript abundance. Taken together, the quantitative real-time PCR assay is standardized, rapid and reproducible for estimation of nicotine conversion level in vivo, which is expected to shed new light on monitoring of burley tobacco converter. PMID:26593897

  6. Quantitative Real-Time PCR Analysis of MicroRNAs and Their Precursors Regulated by TGF-β Signaling.

    PubMed

    Kang, Hara; Hata, Akiko

    2016-01-01

    The signaling pathway of TGF-β and its family member BMP has been implicated in vascular development and maintenance of homeostasis by modulating expression of small noncoding microRNAs (miRNAs). MiRNAs repress target genes, which play a critical role in regulating vascular smooth muscle cell (VSMC) growth, phenotype, and function. To understand the mechanisms by which specific miRNAs control the TGF-β and BMP signaling pathway in VSMC, it is essential to quantitate levels of specific miRNAs and their precursors whose expression are controlled by TGF-β/BMP signaling. Here, we describe a real-time quantization method for accurate and sensitive detection of miRNAs and their precursors, such as primary transcripts of miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs). This method requires two steps; synthesis of single-stranded complementary DNAs (cDNAs) from total RNA samples and quantization of specific pri-, pre-, or mature miRNAs by quantitative polymerase chain reaction (PCR) using a real-time PCR machine.

  7. Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

    PubMed

    Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

    2016-12-01

    This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

  8. Simulation of collaborative studies for real-time PCR-based quantitation methods for genetically modified crops.

    PubMed

    Watanabe, Satoshi; Sawada, Hiroshi; Naito, Shigehiro; Akiyama, Hiroshi; Teshima, Reiko; Furui, Satoshi; Kitta, Kazumi; Hino, Akihiro

    2013-01-01

    To study impacts of various random effects and parameters of collaborative studies on the precision of quantitation methods of genetically modified (GM) crops, we developed a set of random effects models for cycle time values of a standard curve-based relative real-time PCR that makes use of an endogenous gene sequence as the internal standard. The models and data from a published collaborative study for six GM lines at four concentration levels were used to simulate collaborative studies under various conditions. Results suggested that by reducing the numbers of well replications from three to two, and standard levels of endogenous sequence from five to three, the number of unknown samples analyzable on a 96-well PCR plate in routine analyses could be almost doubled, and still the acceptable repeatability RSD (RSDr < or = 25%) and the reproducibility RSD (RSDR < 35%) of the collaborative study could be met. Further, RSDr and RSD(R) were found most sensitive to random effects attributable to inhomogeneity among blind replicates, but they were little influenced by those attributable to DNA extractions. The proposed models are expected to be useful for optimizing standard curve-based relative quantitation methods for GM crops by real-time PCR and their collaborative studies.

  9. Rapid quantitative detection of, Listeria monocytogenes in salmon products: evaluation of pre-real-time PCR strategies.

    PubMed

    Rodríguez-Lázaro, David; Jofré, Anna; Aymerich, Teresa; Garriga, Margarita; Pla, Maria

    2005-07-01

    The spread and persistence of Listeria monocytogenes in smoked fish products and seafood processing factories are big concerns. Thus, the corresponding quality assurance programs must include adequate microbiological control measures. We evaluated eight different pre-PCR sample processing strategies to be coupled with a previously developed real-time PCR assay for the quantitative detection of L. monocytogenes in salmon products. The optimal pre-PCR procedure involved filtration and DNA purification with the use of a commercial kit. This strategy could detect 10 CFU of L. monocytogenes per g of smoked salmon and could quantify 1,000 CFU/g with excellent accuracy compared with the standard plate count method. Thus, this method could be a promising alternative for the quantitative detection of L. monocytogenes in smoked fish products and processing factories. This method could also detect the bacterium in raw salmon.

  10. Modeling real-time PCR kinetics: Richards reparametrized equation for quantitative estimation of European hake (Merluccius merluccius).

    PubMed

    Sánchez, Ana; Vázquez, José A; Quinteiro, Javier; Sotelo, Carmen G

    2013-04-10

    Real-time PCR is the most sensitive method for detection and precise quantification of specific DNA sequences, but it is not usually applied as a quantitative method in seafood. In general, benchmark techniques, mainly cycle threshold (Ct), are the routine method for quantitative estimations, but they are not the most precise approaches for a standard assay. In the present work, amplification data from European hake (Merluccius merluccius) DNA samples were accurately modeled by three sigmoid reparametrized equations, where the lag phase parameter (λc) from the Richards equation with four parameters was demonstrated to be the perfect substitute for Ct for PCR quantification. The concentrations of primers and probes were subsequently optimized by means of that selected kinetic parameter. Finally, the linear correlation among DNA concentration and λc was also confirmed.

  11. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

    PubMed Central

    Expósito-Rodríguez, Marino; Borges, Andrés A; Borges-Pérez, Andrés; Pérez, José A

    2008-01-01

    Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene

  12. Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay.

    PubMed

    Parida, Manmohan; Horioke, Kouhei; Ishida, Hiroyuki; Dash, Paban Kumar; Saxena, Parag; Jana, Asha Mukul; Islam, Mohammed Alimul; Inoue, Shingo; Hosaka, Norimitsu; Morita, Kouichi

    2005-06-01

    The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3' noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.

  13. Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR).

    PubMed

    Marin, Mª José; Figuero, Elena; Herrera, David; Sanz, Mariano

    2017-01-01

    The quantitative polymerase chain reaction (qPCR) is a variant of PCR aimed to detect and quantify a targeted DNA molecule through the addition of probes labeled with fluorescent molecules that emit fluorescence within each amplification cycle, what results in fluorescence values proportional to the amount of accumulated PCR product. This chapter presents the detailed procedures for quantification of different periodontal pathogens (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, and Fusobacterium spp.) using qPCR. It also includes the description of the most frequent problems encountered and how to solve them. In addition, a detailed protocol for multiplex qPCR to detect and quantify P. gingivalis and A. actinomycetemcomitans is included.

  14. Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests.

    PubMed

    Hokynar, Kati; Norja, Päivi; Laitinen, Harri; Palomäki, Pekka; Garbarg-Chenon, Antoine; Ranki, Annamari; Hedman, Klaus; Söderlund-Venermo, Maria

    2004-05-01

    Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.

  15. Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

    PubMed Central

    Naveca, Felipe Gomes; do Nascimento, Valdinete Alves; de Souza, Victor Costa; Nunes, Bruno Tardelli Diniz; Rodrigues, Daniela Sueli Guerreiro; Vasconcelos, Pedro Fernando da Costa

    2017-01-01

    ABSTRACT We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed. PMID:28591313

  16. Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus

    USGS Publications Warehouse

    Pedersen, J.; Killian, M.L.; Hines, N.; Senne, D.; Panigrahy, B.; Ip, H.S.; Spackman, Erica

    2010-01-01

    This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptasePCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 101 50% embryo infectious doses per reaction, or 103104 copies of transcribed H7 RNA. Diagnostic sensitivity and specificity were estimated to be 97.5% and 82.4%, respectively; the assay was shown to be specific for H7 AI when tested with >270 wild birds and poultry viruses. Following validation, the 2008 H7 rRT-PCR procedure was adopted as an official U.S. Department of Agriculture procedure for the detection of H7 AIV. The 2008 H7 assay replaced the previously used (2002) assay, which does not detect H7 viruses currently circulating in wild birds in North and South America. ?? 2010 American Association of Avian Pathologists.

  17. Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses.

    PubMed

    Naveca, Felipe Gomes; Nascimento, Valdinete Alves do; Souza, Victor Costa de; Nunes, Bruno Tardelli Diniz; Rodrigues, Daniela Sueli Guerreiro; Vasconcelos, Pedro Fernando da Costa

    2017-07-01

    We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.

  18. Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR.

    PubMed

    Kuiper, Melanie W; Valster, Rinske M; Wullings, Bart A; Boonstra, Harry; Smidt, Hauke; van der Kooij, Dick

    2006-09-01

    A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.

  19. Epstein-Barr Virus Quantitation by Real-Time PCR Targeting Multiple Gene Segments

    PubMed Central

    Ryan, Julie L.; Fan, Hongxin; Glaser, Sally L.; Schichman, Steven A.; Raab-Traub, Nancy; Gulley, Margaret L.

    2004-01-01

    Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples. PMID:15507678

  20. Nuclear microprobe - synchrotron synergy: towards integrated quantitative real-time elemental imaging using PIXE and SCRF.

    SciTech Connect

    Ryan, C. G.; Etschmann, B. E.; Vogt, S.; Maser, J.; Harland, C. L.; van Achterbergh, E.; Legnini, D.; Experimental Facilities Division; CSIRO Exploration and Mining; Australian Synchrotron Research Program, ANSTO

    2005-01-01

    The Dynamic Analysis (DA) method, for the projection of quantitative elemental images using Proton Induced X-ray Emission (PIXE), has been extended for use with energy-dispersive Synchrotron X-ray Fluorescence (SXRF) data collected with the X-ray microprobe by making use of similarities and synergy with nuclear microscopy. The broad element sensitivity of PIXE is complemented by the selective nature of SXRF, where the beam energy can be tuned to optimize the sensitivity in a portion of the periodic table. PIXE combined with Proton Induced {gamma}-ray Emission (PIGE) in this study provided images of geological samples of 25 elements, including characteristic X-rays up to the energy of the Nd K lines (37 keV). Maximum sensitivity was achieved for elements around Z {approx} 33 with detection limits of {approx}250 ppb (in 5 h). SXRF using a 16.1 keV photon microbeam provided images of 16 elements, with optimum sensitivity around Z {approx} 35 with detection limits of {approx}70 ppb (in 11 h), an improvement of {approx}2.4 times when corrected for acquisition time.

  1. Habitat associations of two entomopathogenic nematodes: a quantitative study using real-time quantitative polymerase chain reactions.

    PubMed

    Torr, Peter; Spiridonov, Sergei E; Heritage, Stuart; Wilson, Michael J

    2007-03-01

    1. Despite nematodes being the most abundant animals on earth, very few animal ecologists study them, probably because of the difficulties of identifying them to species by morphological methods. 2. A group of nematodes that are important both ecologically and economically is the entomopathogenic nematodes, which play a key role in regulating soil food webs and are sold throughout the world as biological insecticides, yet for which very little is known of their population ecology. 3. A novel detection and quantification method was developed for soil nematodes using real-time polymerase chain reaction (PCR), and the technique was used to estimate numbers of two closely related species of entomopathogenic nematodes, Steinernema kraussei and S. affine in 50 soil samples from 10 sites in Scotland representing two distinct habitats (woodland and grassland). 4. There was a high degree of correlation between our molecular and traditional morphological estimates of population size and our data clearly showed that Steinernema affine occurred only in grassland areas, whereas S. kraussei was found in grassland and woodland samples to a similar degree. 5. Real-time PCR offers a rapid and accurate method of detecting individual nematode species from soil samples without the need for a specialist taxonomist, and has much potential for use in studies of nematode population ecology.

  2. A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA.

    PubMed

    Vina-Rodriguez, Ariel; Eiden, Martin; Keller, Markus; Hinrichs, Winfried; Groschup, Martin H

    2016-01-01

    Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

  3. A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA

    PubMed Central

    Vina-Rodriguez, Ariel; Eiden, Martin; Keller, Markus; Hinrichs, Winfried

    2016-01-01

    Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV. PMID:28042576

  4. Real-Time Quantitative Analysis of Valproic Acid in Exhaled Breath by Low Temperature Plasma Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Gong, Xiaoxia; Shi, Songyue; Gamez, Gerardo

    2017-04-01

    Real-time analysis of exhaled human breath is a rapidly growing field in analytical science and has great potential for rapid and noninvasive clinical diagnosis and drug monitoring. In the present study, an LTP-MS method was developed for real-time, in-vivo and quantitative analysis of γ-valprolactone, a metabolite of valproic acid (VPA), in exhaled breath without any sample pretreatment. In particular, the effect of working conditions and geometry of the LTP source on the ions of interest, protonated molecular ion at m/z 143 and ammonium adduct ion at m/z 160, were systematically characterized. Tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) was carried out in order to identify γ-valprolactone molecular ions ( m/z 143), and the key fragment ion ( m/z 97) was used for quantitation. In addition, the fragmentation of ammonium adduct ions to protonated molecular ions was performed in-source to improve the signal-to-noise ratio. At optimum conditions, signal reproducibility with an RSD of 8% was achieved. The concentration of γ-valprolactone in exhaled breath was determined for the first time to be 4.83 (±0.32) ng/L by using standard addition method. Also, a calibration curve was obtained with a linear range from 0.7 to 22.5 ng/L, and the limit of detection was 0.18 ng/L for γ-valprolactone in standard gas samples. Our results show that LTP-MS is a powerful analytical platform with high sensitivity for quantitative analysis of volatile organic compounds in human breath, and can have potential applications in pharmacokinetics or for patient monitoring and treatment.

  5. Application of real-time quantitative polymerase chain reaction assay to detect Legionella pneumophila in patients of community-acquired pneumonia in a tertiary care hospital.

    PubMed

    Angrup, A; Chaudhry, R; Sharma, S; Valavane, A; Passi, K; Padmaja, K; Javed, S; Dey, A B; Dhawan, B; Kabra, S K

    2016-01-01

    Legionella pneumophila is one of the important pathogen responsible for community -acquired pneumonia attributing for 1-5% of cases. Since early and accurate therapy reduces mortality, rapid and reliable diagnostic methods are needed. A total of 134 samples of blood, urine and respiratory tract fluids were collected. Blood was tested for IgG, IgM and IgA antibodies using commercially available kits. A total of 8 (6%) samples were found to be positive for L. pneumophila by quantitative reverse transcription polymerase chain reaction (qRT-PCR), compared to conventional PCR where 6 (4.4%) samples were positive. Serology was positive in a total of 32 (23%) cases though only 3 (2.2%) of the PCR-positive cases were positive by serology as well. These results suggest that real-time PCR can detect Legionella infection early in the course of the disease before serological response develops.

  6. Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

    PubMed

    Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

    2015-10-01

    We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  7. Application of SSH and quantitative real time PCR to construction of gene expression profiles from scallop Chlamys farreri in response to exposure to tetrabromobisphenol A.

    PubMed

    Gong, Xiaoli; Pan, Luqing; Miao, Jingjing; Liu, Na

    2012-11-01

    TBBPA-induced genes were identified using suppression subtractive hybridization (SSH) from Chlamys farreri. A total of 203 and 44 clones from SSH forward and reverse library were respectively obtained including cellular process, immune system process, response to stimulus, metabolic process and signaling etc. Differential gene expressions were compared between scallops from control and TBBPA treatment groups (400 μg/L, 15 days) using quantitative real time RT-PCR. For further research, eight significant genes expression from scallops exposed to TBBPA (0; 100; 200; 400 μg/L) sampling at 0, 1, 3, 6 and 15 days, were utilized for Q-RT-PCR. The results revealed that the expression level of most selected cDNAs was dominantly up-regulated or down-regulated in the TBBPA-induced scallops. These findings provide basic genomic information of the bivalve and the selected genes may be the potential molecular biomarkers for TBBPA pollution in aquatic environment.

  8. Optimization of Quantitative Detection of Cytomegalovirus DNA in Plasma by Real-Time PCR

    PubMed Central

    Boeckh, Michael; Huang, MeeiLi; Ferrenberg, James; Stevens-Ayers, Terry; Stensland, Laurence; Garrett Nichols, W.; Corey, Lawrence

    2004-01-01

    Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log10. Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is

  9. Leptin receptor (LEPR) SNP polymorphisms in HELLP syndrome patients determined by quantitative real-time PCR and melting curve analysis

    PubMed Central

    2010-01-01

    Background Several studies have shown overexpression of leptin in microarray experiments in pre-eclampsia (PE) and in hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. We decided to study four leptin receptor (LEPR) SNP polymorphisms in HELLP syndrome patients by using quantitative real-time PCR and melting curve analysis. Methods DNA was isolated from blood samples from 83 normotensive pregnant women and 75 HELLP syndrome patients. Four SNPs, LEPR c.326A>G (K109), LEPR c.668A>G (Q223R), LEPR c.1968G>C (K656N) and LEPR c.3024A>G (S1008) were determined by quantitative real-time PCR and melting curve analysis. Investigators were blinded to clinical outcomes. Results LEPR c.326A>G, LEPR c.668A>G, LEPR c.1968G>C and LEPR c.3024A>G allele, genotype and haplotype polymorphisms were not different in HELLP syndrome patients and normotensive healthy pregnants. There were strong linkage disequilibrium (LD) between loci c.326A>G and c.6687A>G (D' = 0.974), and c.668A>G and c.1968G>C (D' = 0.934), and c.326A>G and c.1968G>C (D' = 0.885), and c.1968G>C and c.3024A>G (D' = 1.0). However, linkages of c.3024A>G with c.668A>G (D' = 0.111) and c.326A>G (D' = 0.398) were weak. The Hardy-Weinberg equilibrium was observed for all polymorphisms. However the LEPR c.326A>G AG genotype was twice more frequent and the (AG AG GG AG) haplotype was three times more frequent in HELLP syndrome patients. The introduced quantitative real-time PCR combined with melting curve analysis is a fast and reliable method for the determination of LEPR SNPs. Conclusion Although certain LEPR haplotypes are more frequent in HELLP syndrome, we conclude that there is no compelling evidence that the four studied LEPR SNP polymorphisms associated with the development of HELLP syndrome. PMID:20149225

  10. Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

    USGS Publications Warehouse

    Chase, D.M.; Elliott, D.G.; Pascho, R.J.

    2006-01-01

    Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

  11. Real-time PCR for quantitative detection of chamois (Rupicapra rupicapra) and pyrenean ibex (Capra pyrenaica) in meat mixtures.

    PubMed

    Fajardo, Violeta; González, Isabel; Martín, Irene; Rojas, María; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2008-01-01

    A real-time quantitative polymerase chain reaction (PCR) technique was developed for the quantification of chamois and pyrenean ibex DNAs in meat mixtures by using a SYBR green detection platform. Two species-specific systems and a eukaryotic endogenous system were combined in the real-time PCR approach to quantify the target species. In the specific systems, a 133 base pair (bp) fragment of the 12S rRNA gene was amplified from chamois DNA, and an 88 bp fragment from the D-loop region was amplified from pyrenean ibex DNA. In the endogenous system, universal primers amplified a 141 bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The threshold cycle values obtained with the 18S rRNA primers were used to normalize those obtained from chamois- or pyrenean ibex-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat-treated binary mixtures of chamois and pyrenean ibex meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target DNAs in the range of 0.1-0.8%, depending on the species and treatment of the meat samples.

  12. Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.

    PubMed

    Watzinger, F; Suda, M; Preuner, S; Baumgartinger, R; Ebner, K; Baskova, L; Niesters, H G M; Lawitschka, A; Lion, T

    2004-11-01

    A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.

  13. Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay.

    PubMed

    Kao, Po-Min; Tung, Min-Che; Hsu, Bing-Mu; Chiu, Yi-Chou; She, Cheng-Yu; Shen, Shu-Min; Huang, Yu-Li; Huang, Wen-Chien

    2013-09-01

    In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6 % (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6 %, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75 × 10(4) and 3.47 × 10(5) cells/L in river water, 6.92 × 10(4) and 4.29 × 10(5) cells/L in raw drinking water, and 5.71 × 10(4) and 2.12 × 10(6) cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8 %), Legionella jordanis (4.2 %), Legionella nautarum (4.2 %), Legionella sp. (4.2 %), and uncultured Legionella sp. (66.6 %). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.

  14. Quantitative real-time polymerase chain reaction for malaria diagnosis and its use in malaria vaccine clinical trials.

    PubMed

    Andrews, Laura; Andersen, Rikke F; Webster, Daniel; Dunachie, Susanna; Walther, R Michael; Bejon, Philip; Hunt-Cooke, Angela; Bergson, Gillian; Sanderson, Frances; Hill, Adrian V S; Gilbert, Sarah C

    2005-07-01

    The demand for an effective malaria vaccine is high, with millions of people being affected by the disease every year. A large variety of potential vaccines are under investigation worldwide, and when tested in clinical trials, researchers need to extract as much data as possible from every vaccinated and control volunteer. The use of quantitative real-time polymerase chain reaction (PCR), carried out in real-time during the clinical trials of vaccines designed to act against the liver stage of the parasite's life cycle, provides more information than the gold standard method of microscopy alone and increases both safety and accuracy. PCR can detect malaria parasites in the blood up to 5 days before experienced microscopists see parasites on blood films, with a sensitivity of 20 parasites/mL blood. This PCR method has so far been used to follow 137 vaccinee and control volunteers in Phase IIa trials in Oxford and on 220 volunteer samples during a Phase IIb field trial in The Gambia.

  15. Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

    PubMed

    Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

    2009-08-01

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  16. Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle

    PubMed Central

    2012-01-01

    Background The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered. Results We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca. Conclusions The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures. PMID:22889141

  17. A Bayesian method for calculating real-time quantitative PCR calibration curves using absolute plasmid DNA standards.

    PubMed

    Sivaganesan, Mano; Seifring, Shawn; Varma, Manju; Haugland, Richard A; Shanks, Orin C

    2008-02-25

    In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards. Instead of the two traditional methods (classical and inverse), a Monte Carlo Markov Chain (MCMC) estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest. The Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards.

  18. In-house quantitative real-time PCR for the diagnosis of hepatitis B virus and hepatitis C virus infections.

    PubMed

    Zauli, Danielle Alves Gomes; Menezes, Carla Lisandre Paula de; Oliveira, Cristiane Lommez de; Mateo, Elvis Cristian Cueva; Ferreira, Alessandro Clayton de Souza

    The quantification of viral nucleic acids in serum by real-time PCR plays an important role in diagnosing hepatitis B virus and hepatitis C virus infection. In this study, we developed an assay using specific primers and probes to quantify hepatitis B virus DNA or hepatitis C virus RNA in serum from infected patients. For standardization and validation of the assay, an international panel of hepatitis B virus/hepatitis C virus and standard plasmids was used. A correlation coefficient of 0.983 and 0.963 for hepatitis B virus and hepatitis C virus, respectively, was obtained based on cycle threshold values and concentrations of DNA or RNA. The standard curve showed a linear relationship from 19IU/mL to 1.9×10(9)IU/mL of serum, with a coefficient of determination (r(2)) of 0.99. In sera from patients infected with hepatitis B virus or hepatitis C virus viral loads (19IU/mL and 1.9×10(9)IU/mL), we quantified viral loads with a detection limit of 1.9×10(2)IU/mL. The real-time quantitative PCR assay developed in this study provides an ideal system for routine diagnosis and confirmation of indeterminate serological results, especially in immunosuppressed patients. Copyright © 2016. Published by Elsevier Editora Ltda.

  19. Quantitative assessment of minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction.

    PubMed

    Yamazaki, Jumpei; Baba, Kenji; Goto-Koshino, Yuko; Setoguchi-Mukai, Asuka; Fujino, Yasuhito; Ohno, Koichi; Tsujimoto, Hajime

    2008-12-15

    Lymphoma is the most common hematopoietic malignancy in dogs. Although a large proportion of dogs with lymphoma can achieve clinical remission by initial chemotherapy, most dogs die as a consequence of tumor relapse. We established a quantitative detection system for minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction (PCR). A canine T-cell lymphoma-derived cell line, namely, UL-1, was used to examine the specificity and sensitivity of the MRD detecting system. Allele-specific oligonucleotide primers and probes were designed based on the sequence of T-cell receptor gamma chain (TCRgamma) gene fragment of UL-1 cells in conjunction with its downstream sequence, which were obtained from the dog genome database. The real-time PCR system for plasmid DNA containing the TCRgamma gene derived from UL-1 cells and the genomic DNA of UL-1 cells revealed that the system was accurate for 10-100,000 copies per reaction and its sensitivity was 1 cell per 10,000 cells. In order to monitor the kinetics of tumor cell number in canine lymphoma, we quantified the level of MRD in the peripheral blood of 7 dogs with lymphoma under chemotherapy. Since the lymphoma cells from the 7 patients were shown to be B-cell origin from the finding of clonal rearrangement of immunoglobulin heavy chain (IgH) gene, allele-specific oligonucleotide primers and probes were prepared based on the sequence of rearranged IgH gene in each case. The number of peripheral blood tumor cells measured by the real-time PCR was comparable to that estimated by conventional hematological examination in 2 cases of stage V lymphoma. MRD in the peripheral blood was detectable in all 7 cases, even in the complete remission (CR) phase. In the 7 lymphoma dogs, changes in the MRD levels of peripheral blood generally paralleled with the changes in the volumes of lymph nodes. Molecular CR, in which the MRD level was below the detection limit, was not observed in any of these 7 patients

  20. A One-Step Real-Time RT-PCR Assay for the Detection and Quantitation of Sugarcane Streak Mosaic Virus.

    PubMed

    Fu, Wei-Lin; Sun, Sheng-Ren; Fu, Hua-Ying; Chen, Ru-Kai; Su, Jin-Wei; Gao, San-Ji

    2015-01-01

    Sugarcane mosaic disease is caused by the Sugarcane streak mosaic virus (SCSMV; genus Poacevirus, family Potyviridae) which is common in some Asian countries. Here, we established a protocol of a one-step real-time quantitative reverse transcription PCR (real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane. Primers and probes were designed within the conserved region of the SCSMV coat protein (CP) gene sequences. Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmid in vitro and total RNA extracted from SCSMV-infected sugarcane were used as templates of qRT-PCR. We further performed a sensitivity assay to show that the detection limit of the assay was 100 copies of ssRNA and 2 pg of total RNA with good reproducibility. The values obtained were approximately 100-fold more sensitive than those of the conventional RT-PCR. A higher incidence (68.6%) of SCSMV infection was detected by qRT-PCR than that (48.6%) with conventional RT-PCR in samples showing mosaic symptoms. SCSMV-free samples were verified by infection with Sugarcane mosaic virus (SCMV) or Sorghum mosaic virus (SrMV) or a combination of both. The developed qRT-PCR assay may become an alternative molecular tool for an economical, rapid, and efficient detection and quantification of SCSMV.

  1. Fast Quantitative Real-Time PCR-Based Screening for Common Chromosomal Aneuploidies in Mouse Embryonic Stem Cells

    PubMed Central

    D’Hulst, Charlotte; Parvanova, Irena; Tomoiaga, Delia; Sapar, Maria L.; Feinstein, Paul

    2013-01-01

    Summary Chromosomal integrity has been known for many years to affect the ability of mouse embryonic stem cells (mESCs) to contribute to the germline of chimeric mice. Abnormal chromosomes are generally detected by standard cytogenetic karyotyping. However, this method is expensive, time consuming, and often omitted prior to blastocyst injection, consequently reducing the frequency of mESC-derived offspring. Here, we show a fast, accurate, and inexpensive screen for identifying the two most common aneuploidies (Trisomy 8 and loss of chromosome Y) in genetically manipulated mESCs using quantitative real-time PCR (qPCR). Screening against these two aneuploidies significantly increases the fraction of normal mESC clones. Our method is extremely sensitive and can detect as low as 10% aneuploidy among a large population of mESCs. It greatly expedites the generation of mutant mice and provides a quick tool for assessing the aneuploidy percentages of any mESC line. PMID:24319669

  2. Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR).

    PubMed

    Forootan, Amin; Sjöback, Robert; Björkman, Jens; Sjögreen, Björn; Linz, Lucas; Kubista, Mikael

    2017-06-01

    Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

  3. Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.

    PubMed

    Tobias, T J; Bouma, A; Klinkenberg, D; Daemen, A J J M; Stegeman, J A; Wagenaar, J A; Duim, B

    2012-08-01

    A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

    PubMed

    Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

    2016-01-01

    This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.

  5. SHORT-COMMUNICATION Validation of reference genes for real-time quantitative PCR in tambaqui (Colossoma macropomum).

    PubMed

    Nascimento, A R; Silva, G F; Gualberto, G F; Almeida, F L

    2016-12-23

    Tambaqui, Colossoma macropomum, is the main native freshwater fish in Brazilian aquaculture. Therefore, intensive research pressure has been applied to the species to support new technologies for tambaqui farming. Molecular biology represents a tool that can be used to investigate every field of applied biology, from fish physiology to the effects of climate change. Based on the importance of reference genes for the relative or absolute quantification of gene transcripts, we cloned and sequenced three candidate reference genes in tambaqui (18S ribossomal RNA - 18s, glyceraldehyde-3-phosphate dehydrogenase - gapdh, and actin beta - β-actin), and validated a set of primers for each gene for use in real-time quantitative PCR. The results were evaluated by RefFinder, which indicated that β-actin is the most suitable reference gene for tambaqui among those studied, followed by 18s.

  6. Real-time molecular profiling of photochemically induced rat thrombosis in vivo through quantitative Raman analysis of blood

    NASA Astrophysics Data System (ADS)

    Lin, M. M.; Shen, A. G.; Yao, H. L.; Zhang, Z. Z.; Hu, J. M.

    2014-11-01

    A device of an animal thrombosis model in vivo coupled with a Raman system for near-surface blood vessels is proposed in this letter. The dual-function set up is capable of simultaneously establishing a photochemically induced artificial thrombus model and collecting in vivo Raman data of both arterial and venous blood, and it provides the first observation of rat thrombosis under the physiological conditions from the beginning to the final form. The real-time and quantitative molecular profiling of flowing blood and the spectra of blood cells in the process of thrombosis provides an insight into the occurring mechanism of thrombosis and a promising method for the in vivo screening of new antithrombotic and thrombolytic drugs.

  7. Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

    PubMed

    Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

    2010-12-01

    The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

  8. Standardisation of data from real-time quantitative PCR methods – evaluation of outliers and comparison of calibration curves

    PubMed Central

    Burns, Malcolm J; Nixon, Gavin J; Foy, Carole A; Harris, Neil

    2005-01-01

    Background As real-time quantitative PCR (RT-QPCR) is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR technique, through investigation of the experimental design and operating procedure. However, measurement uncertainty contributions made during the data analysis procedure have not been studied in detail. This paper presents two additional approaches for standardising data analysis through the novel application of statistical methods to RT-QPCR, in order to minimise potential uncertainty in results. Results Experimental data was generated in order to develop the two aspects of data handling and analysis that can contribute towards measurement uncertainty in results. This paper describes preliminary aspects in standardising data through the application of statistical techniques to the area of RT-QPCR. The first aspect concerns the statistical identification and subsequent handling of outlying values arising from RT-QPCR, and discusses the implementation of ISO guidelines in relation to acceptance or rejection of outlying values. The second aspect relates to the development of an objective statistical test for the comparison of calibration curves. Conclusion The preliminary statistical tests for outlying values and comparisons between calibration curves can be applied using basic functions found in standard spreadsheet software. These two aspects emphasise that the comparability of results arising from RT-QPCR needs further refinement and development at the data-handling phase. The implementation of standardised approaches to data analysis should further help minimise variation due to subjective judgements. The aspects described in this paper will help contribute towards the

  9. Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue.

    PubMed

    Zhang, Wan-Xia; Fan, Jie; Ma, Jing; Rao, Yi-Song; Zhang, Li; Yan, You-E

    2016-06-22

    Quantitative real-time PCR (qRT-PCR) is the most classical technique in the field of gene expression study. This method requires an appropriate reference gene to normalize mRNA levels. In this study, the expression stability of four frequently-used reference genes in epididymal white adipose tissue (eWAT), inguinal beige adipose tissue (iBeAT) and brown adipose tissue (BAT) from obese and lean rats were evaluated by geNorm, NormFinder and BestKeeper. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the two most stable reference genes were recommended in each type of adipose tissue. Two target genes were applied to test the stability of the reference genes. The geNorm and NormFinder results revealed that GAPDH and 36B4 exhibited the highest expression stabilities in eWAT, while 36B4 and β-actin had the highest expression stabilities in iBeAT and BAT. According to the results of the BestKeeper analysis, 36B4 was the most stable gene in eWAT, iBeAT and BAT, in terms of the coefficient of variance. In terms of the coefficient of correlation, GAPDH, 36B4 and β-actin were the most stable genes in eWAT, iBeAT and BAT, respectively. Additionally, expected results and statistical significance were obtained using a combination of two suitable reference genes for data normalization. In conclusion, 36B4 and GAPDH, in combination, are the best reference genes for eWAT, while 36B4 and β-actin are two most suitable reference genes for both iBeAT and BAT. We recommend using these reference genes accordingly.

  10. Evaluation of normalization strategies used in real-time quantitative PCR experiments in HepaRG cell line studies.

    PubMed

    Ceelen, Liesbeth; De Craene, Jurgen; De Spiegelaere, Ward

    2014-03-01

    The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored. In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.

  11. Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays.

    PubMed

    Kolb, Steffen; Knief, Claudia; Stubner, Stephan; Conrad, Ralf

    2003-05-01

    Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the

  12. Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes.

    PubMed

    Petz, Lawrence N; Turell, Michael J; Padilla, Susana; Long, Lewis S; Reinbold-Wasson, Drew D; Smith, Darci R; O'Guinn, Monica L; Melanson, Vanessa R; Lee, John S

    2014-10-01

    Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens. © The American Society of Tropical Medicine and Hygiene.

  13. Real-time reverse transcriptase PCR for the rapid and sensitive detection of Salmonella typhimurium from pork.

    PubMed

    Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

    2010-03-01

    Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 10(8) to 10(0) CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37 degrees C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (T(m)) analysis to determine specific Salmonella invA (T(m) = 87.5 degrees C) and IAC (T(m) = 82 degrees C) products. Improved Salmonella detection up to 10(1) CFU/25 g of pork and 10(0) CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 10(6) CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take >/=1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.

  14. Development, optimization, and validation of a Classical swine fever virus real-time reverse transcription polymerase chain reaction assay.

    PubMed

    Eberling, August J; Bieker-Stefanelli, Jill; Reising, Monica M; Siev, David; Martin, Barbara M; McIntosh, Michael T; Beckham, Tammy R

    2011-09-01

    Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a well-characterized assay that can deliver a rapid, accurate diagnosis prior to the onset of clinical signs. A real-time fluorogenic-probe hydrolysis (TaqMan) reverse transcription polymerase chain reaction (RT-PCR) for CSF was developed by the United States Department of Agriculture (USDA) at the Plum Island Animal Disease Center (CSF PIADC assay) and evaluated for analytical and diagnostic sensitivity and specificity. A well-characterized panel including Classical swine fever virus (CSFV), Bovine viral diarrhea virus (BVDV), and Border disease virus (BDV) isolates was utilized in initial feasibility and optimization studies. The assay was initially designed and validated for use on the ABI 7900HT using the Qiagen QuantiTect® Probe RT-PCR chemistry. However, demonstrating equivalency with multiple one-step RT-PCR chemistries and PCR platforms increased the versatility of the assay. Limit of detection experiments indicated that the Qiagen QuantiTect® Multiplex (NoROX) and the Invitrogen SuperScript® III RT-PCR kits were consistently the most sensitive one-step chemistries for use with the CSF PIADC primer/probe set. Analytical sensitivity of the CSF PIADC assay ranged from <1-2.95 log(10) TCID(50)/ml on both the ABI 7900HT and ABI 7500 platforms. The CSF PIADC assay had 100% diagnostic sensitivity and specificity when tested on a panel of 152 clinical samples from the Dominican Republic and Colombia. The ability to perform this newly developed assay in 96-well formats provides an increased level of versatility for use in CSF surveillance programs.

  15. Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection

    PubMed Central

    Lavenir, Rachel; Lepelletier, Anthony; Faouzi, Abdellah; Troupin, Cécile; Nourlil, Jalal; Buchy, Philippe; Bourhy, Herve

    2016-01-01

    The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus

  16. Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

    PubMed

    Dacheux, Laurent; Larrous, Florence; Lavenir, Rachel; Lepelletier, Anthony; Faouzi, Abdellah; Troupin, Cécile; Nourlil, Jalal; Buchy, Philippe; Bourhy, Herve

    2016-07-01

    The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus

  17. [Detection of hematopoietic chimera by real-time fluorescent quantitative PCR with erythrocyte Kidd blood group gene].

    PubMed

    Chen, Shu; Xu, Xian-Guo; Liu, Ying; Hong, Xiao-Zhen; Zhu, Fa-Ming; Lü, Hang-Jun; Yan, Li-Xing

    2012-06-01

    This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.

  18. Comparison of droplet digital PCR and quantitative real-time PCR in mcrA-based methanogen community analysis.

    PubMed

    Kim, Tae Gwan; Jeong, So-Yeon; Cho, Kyung-Suk

    2014-12-01

    Two different quantitative PCR platforms, droplet digital PCR (dd-PCR) and quantitative real-time PCR (qPCR), were compared in a mcrA-based methanogen community assay that quantifies ten methanogen sub-groups. Both technologies exhibited similar PCR efficiencies over at least four orders of magnitude and the same lower limits of detection (8 copies μL-DNA extract(-1)). The mcrA-based methanogen communities in three full-scale anaerobic digesters were examined using the two technologies. dd-PCR detected seven groups from the digesters, while qPCR did five groups, indicating that dd-PCR is more sensitive for DNA quantification. Linear regression showed quantitative agreements between both of the technologies (R(2) = 0.59-0.98) in the five groups that were concurrently detected. Principal component analysis from the two datasets consistently indicated a substantial difference in the community composition among the digesters and revealed similar levels of differentiation among the communities. The combined results suggest that dd-PCR is more promising for examining methanogenic archaeal communities in biotechnological processes.

  19. Semi-quantitative real-time PCR: A useful approach to identify persons with low replicative chronic hepatitis B.

    PubMed

    Castéra-Guy, Joany; Rubbo, Pierre-Alain; Kania, Dramane; Lemoine, Maud; Van de Perre, Philippe; Tuaillon, Edouard

    2017-06-01

    Antiviral therapy can be avoided during the low replicative phase of chronic Hepatitis B virus (HBV) infection which is characterized notably by HBV DNA concentration below 2000IU/ml. Simplified diagnostic tests can improve access to HBV DNA monitoring in resource-limited settings. The capacity of a new semi-quantitative real-time PCR approach based on sample-to-standard relative detection of the target to discriminate samples with HBV DNA levels above or below the clinical threshold of 2000IU/ml was compared to a quantitative assay (Roche CobasAmpliPrep/CobasTaqMan HBV Test v2.0). The semi-quantitative assay correctly identified 40/40 (100%) low replicative HBV DNA patients and 58/61 (95%) samples from HBV-infected subjects with moderate/high levels of viral DNA. Our results suggested that this alternative PCR test is efficient to guide therapeutic decision based on identification of low replicative HBV infection from all of the chronic hepatitis B carriers requiring treatment, and may be useful in resource-limited settings where the vast majority of cases live. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Real-Time Quantitative Analysis of H2, He, O2, and Ar by Quadrupole Ion Trap Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Ottens, Andrew K.; Harrison, W. W.; Griffin, Timothy P.; Helms, William R.; Voska, N. (Technical Monitor)

    2002-01-01

    The use of a quadrupole ion trap mass spectrometer for quantitative analysis of hydrogen and helium as well as other permanent gases is demonstrated. The customized instrument utilizes the mass selective instability mode of mass analysis as with commercial instruments; however, this instrument operates at a greater RF trapping frequency and without a buffer gas. With these differences, a useable mass range from 2 to over 50 Da is achieved, as required by NASA for monitoring the Space Shuttle during a launch countdown. The performance of the ion trap is evaluated using part-per-million concentrations of hydrogen, helium, oxygen and argon mixed into a nitrogen gas stream. Relative accuracy and precision when quantitating the four analytes were better than the NASA-required minimum of 10% error and 5% deviation, respectively. Limits of detection were below the NASA requirement of 25-ppm hydrogen and 100-ppm helium; those for oxygen and argon were slightly higher than the requirement. The instrument provided adequate performance at fast data recording rates, demonstrating the utility of an ion trap mass spectrometer as a real-time quantitative monitoring device for permanent gas analysis.

  1. Real-Time Quantitative Analysis of H2, He, O2, and Ar by Quadrupole Ion Trap Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Ottens, Andrew K.; Harrison, W. W.; Griffin, Timothy P.; Helms, William R.; Voska, N. (Technical Monitor)

    2002-01-01

    The use of a quadrupole ion trap mass spectrometer for quantitative analysis of hydrogen and helium as well as other permanent gases is demonstrated. The customized instrument utilizes the mass selective instability mode of mass analysis as with commercial instruments; however, this instrument operates at a greater RF trapping frequency and without a buffer gas. With these differences, a useable mass range from 2 to over 50 Da is achieved, as required by NASA for monitoring the Space Shuttle during a launch countdown. The performance of the ion trap is evaluated using part-per-million concentrations of hydrogen, helium, oxygen and argon mixed into a nitrogen gas stream. Relative accuracy and precision when quantitating the four analytes were better than the NASA-required minimum of 10% error and 5% deviation, respectively. Limits of detection were below the NASA requirement of 25-ppm hydrogen and 100-ppm helium; those for oxygen and argon were slightly higher than the requirement. The instrument provided adequate performance at fast data recording rates, demonstrating the utility of an ion trap mass spectrometer as a real-time quantitative monitoring device for permanent gas analysis.

  2. Comparison between Culture and a Multiplex Quantitative Real-Time Polymerase Chain Reaction Assay Detecting Ureaplasma urealyticum and U. parvum

    PubMed Central

    Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

    2014-01-01

    A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR. PMID:25047036

  3. Passive Fourier-transform infrared spectroscopy of chemical plumes: an algorithm for quantitative interpretation and real-time background removal

    NASA Astrophysics Data System (ADS)

    Polak, Mark L.; Hall, Jeffrey L.; Herr, Kenneth C.

    1995-08-01

    We present a ratioing algorithm for quantitative analysis of the passive Fourier-transform infrared spectrum of a chemical plume. We show that the transmission of a near-field plume is given by tau plume = (Lobsd - Lbb-plume)/(Lbkgd - Lbb-plume), where tau plume is the frequency-dependent transmission of the plume, L obsd is the spectral radiance of the scene that contains the plume, Lbkgd is the spectral radiance of the same scene without the plume, and Lbb-plume is the spectral radiance of a blackbody at the plume temperature. The algorithm simultaneously achieves background removal, elimination of the spectrometer internal signature, and quantification of the plume spectral transmission. It has applications to both real-time processing for plume visualization and quantitative measurements of plume column densities. The plume temperature (Lbb-plume ), which is not always precisely known, can have a profound effect on the quantitative interpretation of the algorithm and is discussed in detail. Finally, we provide an illustrative example of the use of the algorithm on a trichloroethylene and acetone plume.

  4. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    PubMed

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2017-08-29

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  5. A Capsid Gene-Based Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Detection of Marine Vesiviruses in the Caliciviridae

    USDA-ARS?s Scientific Manuscript database

    A real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was developed for the identification of marine vesiviruses. The primers were designed to target a 176-nucleotide fragment within a highly conserved region of the San Miguel sea lion viruses (SMSVs) capsid gene. The assay de...

  6. Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza Virus by Real-Time Reverse Transcription Polymerase Chain Reaction Assays

    USDA-ARS?s Scientific Manuscript database

    A multiplex Taqman®-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to detect all strains of Citrus tristeza virus (CTV) and to identify potentially severe strains of the virus. A CTV TaqMan probe (CTV-CY5) based on the coat protein (CP) gene sequences...

  7. Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae).

    EPA Science Inventory

    A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assa...

  8. Specific detection of rinderpest virus by real-time reverse transcription-PCR in preclincal and clinical samples of experimentally infected cattle

    USDA-ARS?s Scientific Manuscript database

    A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (RT-PR) system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detec...

  9. Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae).

    EPA Science Inventory

    A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assa...

  10. Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

    PubMed

    Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

    2009-06-01

    Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

  11. Quantitative assessment of tumor angiogenesis using real-time motion-compensated contrast-enhanced ultrasound imaging

    PubMed Central

    Pysz, Marybeth A.; Guracar, Ismayil; Foygel, Kira; Tian, Lu; Willmann, Jürgen K.

    2015-01-01

    Purpose To develop and test a real-time motion compensation algorithm for contrast-enhanced ultrasound imaging of tumor angiogenesis on a clinical ultrasound system. Materials and methods The Administrative Institutional Panel on Laboratory Animal Care approved all experiments. A new motion correction algorithm measuring the sum of absolute differences in pixel displacements within a designated tracking box was implemented in a clinical ultrasound machine. In vivo angiogenesis measurements (expressed as percent contrast area) with and without motion compensated maximum intensity persistence (MIP) ultrasound imaging were analyzed in human colon cancer xenografts (n = 64) in mice. Differences in MIP ultrasound imaging signal with and without motion compensation were compared and correlated with displacements in x- and y-directions. The algorithm was tested in an additional twelve colon cancer xenograft-bearing mice with (n = 6) and without (n = 6) anti-vascular therapy (ASA-404). In vivo MIP percent contrast area measurements were quantitatively correlated with ex vivo microvessel density (MVD) analysis. Results MIP percent contrast area was significantly different (P < 0.001) with and without motion compensation. Differences in percent contrast area correlated significantly (P < 0.001) with x- and y-displacements. MIP percent contrast area measurements were more reproducible with motion compensation (ICC = 0.69) than without (ICC = 0.51) on two consecutive ultrasound scans. Following anti-vascular therapy, motion-compensated MIP percent contrast area significantly (P = 0.03) decreased by 39.4 ± 14.6 % compared to non-treated mice and correlated well with ex vivo MVD analysis (Rho = 0.70; P = 0.05). Conclusion Real-time motion-compensated MIP ultrasound imaging allows reliable and accurate quantification and monitoring of angiogenesis in tumors exposed to breathing-induced motion artifacts. PMID:22535383

  12. A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus.

    PubMed

    Zeschnigk, Michael; Böhringer, Stefan; Price, Elizabeth Ann; Onadim, Zerrin; Masshöfer, Lars; Lohmann, Dietmar R

    2004-09-07

    Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.

  13. Rapid and direct quantitative detection of viable bifidobacteria in probiotic yogurt by combination of ethidium monoazide and real-time PCR using a molecular beacon approach.

    PubMed

    Meng, X C; Pang, R; Wang, C; Wang, L Q

    2010-11-01

    The potential of ethidium monoazide (EMA) real-time PCR method based on molecular beacon probe for rapid detection of viable bifidobacteria present in probiotic yogurt was evaluated in this work. A real-time PCR with molecular beacon assay was developed to determine genus Bifidobacterium quantitatively in order to increase the sensitivity and specificity of assay. EMA was used to treat probiotic yogurt prior to DNA extraction and real-time PCR detection to allow detection of only viable bacteria. The primer set of Bif-F/Bif-R which is genus-specific for Bifid. was designed. The specificity of the probes ensures that no signal is generated by non-target amplicons. Linear regression analysis demonstrated a good correlation (R² = 0·9948) between the EMA real-time PCR results and the plate counting, and real-time quantitative PCR results correlated adequately with enumeration of bifidobacteria by culture for commercial probiotic yogurt. This culture-independent approach is promising for the direct and rapid detection of viable bifidobacteria in commercial probiotic yogurt, and the detection can be carried out within 4 h. The detection limit for this method is about 10⁴ cell/ml. In conclusion, the direct quantitative EMA real-time PCR assay based on molecular beacon described in this research is a rapid and quantitative method.

  14. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

    PubMed Central

    Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

    2009-01-01

    Background Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. Results From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. Conclusion In this work, we provided a set of genes that are suitable reference

  15. Quantitative real-time PCR (qPCR) for Eimeria tenella replication — Implications for experimental refinement and animal welfare

    PubMed Central

    Nolan, Matthew J.; Tomley, Fiona M.; Kaiser, Pete; Blake, Damer P.

    2015-01-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings

  16. Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.

    PubMed

    Nolan, Matthew J; Tomley, Fiona M; Kaiser, Pete; Blake, Damer P

    2015-10-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R(2)=0.994) (p<0.002) but not in those from day eight (after most oocyst shedding) (R(2)=0.006) (p>0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R(2)=0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings.

  17. Diagnostic value of a "wide-range" quantitative nested real-time PCR assay for varicella zoster virus myelitis.

    PubMed

    Takahashi, Teruyuki; Tamura, Masato; Takasu, Toshiaki

    2013-11-01

    Myelitis is one of the rarest neurological complications of varicella zoster virus (VZV) infection. In this study, the authors remodeled the "wide-range" quantitative nested real-time (QNRT) polymerase chain reaction (PCR) assay to quantitatively detect a small amount of VZV-DNA in cerebrospinal fluid (CSF). For use as a specific internal control "calibrator," an original mutation-VZV (MZ) plasmid was developed. The initial copy number of VZV-DNA in CSF specimens was measured by the amplification rate of the MZ-plasmid. For 17 consecutive CSF specimens collected from three elderly patients with VZV myelitis, the diagnostic value of the wide-range QNRT-PCR assay was evaluated and compared with other conventional PCR assays and enzyme immunoassay (EIA). The MZ-plasmid demonstrated statistically uniform amplifications (F=1.016) against a wide range (1-100,000) of copy numbers of mimic VZV-DNA. The wide-range QNRT-PCR assay quantitatively and rapidly (within 48 hr) detected 5,863, 3,052, 958, and 6,721 copies/ml of VZV-DNA in the CSF specimens collected from all patients in the acute phase. Additionally, there was a significant difference (*P=0.023) in the copy number of VZV-DNA between before and after acyclovir treatment. Other conventional single PCR assays all revealed negative results, but were nevertheless time-consuming (7 days). The IgG EIA-value for VZV was continually elevated throughout the clinical course of all patients. The MZ-plasmid was thus regarded as an appropriate "calibrator" in the wide-range QNRT-PCR assay. This assay is a novel, rapid, accurate, quantitative, and highly sensitive technique, and will contribute as a reliable and useful clinical examination for the rapid diagnosis of VZV infection to central nervous system. © 2013 Wiley Periodicals, Inc.

  18. A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

    PubMed

    Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

    2014-01-01

    Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes.

  19. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    PubMed

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

  20. Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

    PubMed Central

    Smith, William L.; Chadwick, Sean G.; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E.; Aguin, Tina J.; Sobel, Jack D.

    2016-01-01

    Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV. PMID:26818677

  1. Quantitative Real-Time Measurements of DNA Hybridization with Alkylated Non-Oxidized Silicon Nanowires in Electrolyte Solution

    PubMed Central

    Bunimovich, Yuri L.; Shin, Young Shik; Yeo, Woon-Seok; Amori, Michael; Kwong, Gabriel

    2013-01-01

    The quantitative, real time detection of single stranded oligonucleotides with silicon nanowires (SiNWs) in physiologically relevant electrolyte solution is demonstrated. Debye screening of the hybridization event is minimized by utilizing electrostatically adsorbed primary DNA on an amine-terminated NW surface. Two surface functionalization chemistries are compared: an amine terminated siloxane monolayer on the native SiO2 surface of the SiNW, and an amine terminated alkyl monolayer grown directly on a hydrogen-terminated SiNW surface. The SiNWs without the native oxide exhibit improved solution-gated field-effect transistor characteristics and a significantly enhanced sensitivity to single stranded DNA detection, with an accompanying two orders of magnitude improvement in the dynamic range of sensing. A model for the detection of analyte by SiNW sensors is developed and utilized to extract DNA binding kinetic parameters. Those values are directly compared with values obtained by the standard method of surface plasmon resonance (SPR), and demonstrated to be similar. The nanowires, however, are characterized by higher detection sensitivity. The implication is that Si NWs can be utilized to quantitate the solution phase concentration of biomolecules at low concentrations. This work also demonstrates the importance of surface chemistry for optimizing biomolecular sensing with silicon nanowires. PMID:17165787

  2. Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

    PubMed

    Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

    2013-10-10

    Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots.

  3. Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples.

    PubMed

    Gokduman, Kurtulus; Avsaroglu, M Dilek; Cakiris, Aris; Ustek, Duran; Gurakan, G Candan

    2016-03-01

    The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 10(1)CFU/ml and 10(0)CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I--The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II--The method is applicable to challenging samples, such as milk; III--The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves.

  4. A Quantitative Real-Time PCR Approach for Assessing Campylobacter jejuni and Campylobacter coli Colonization in Broiler Herds.

    PubMed

    Haas, Katrin; Overesch, Gudrun; Kuhnert, Peter

    2017-04-01

    Human campylobacteriosis is a major public health concern in developed countries, with Campylobacter jejuni and Campylobacter coli from poultry recognized as the main source of human infection. Identification of Campylobacter-positive broiler herds before slaughter is essential for implementing measures to avoid carryover of pathogens via the slaughter process into the food chain. However, appropriate methods that have been validated for testing poultry flocks antemortem are lacking for Campylobacter. A quantitative real-time PCR (qPCR) that allows simultaneous detection and quantification of C. jejuni and C. coli was adapted and optimized to be applied on boot socks. The adjusted qPCR serves as an easy, sensitive, and quantitative method for Campylobacter detection in poultry flocks antemortem by analysis of boot socks. An adequate correlation was found between qPCR and culture, as well as between boot socks and cecal samples, which are regarded as the "gold standard." Therefore, boot sock sampling followed by qPCR analysis provides a reliable and simple method for assessing Campylobacter load within a flock prior to slaughter. The approach allows categorization of broiler herds into negative, low, moderate, or high Campylobacter colonization. Based on the results of this new approach, risk assessment models, such as evaluating the possible effect of sorting flocks before slaughter, can be easily implemented. Similarly, targeted identification of highly colonized flocks for improvement of biosecurity measures at the farm level will become feasible, presenting an opportunity to increase food safety.

  5. A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

    PubMed Central

    Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

    2008-01-01

    Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost. PMID:18522756

  6. Real-time reverse-transcriptase polymerase chain reaction for the rapid detection of Salmonella using invA primers.

    PubMed

    D'Souza, Doris H; Critzer, Faith J; Golden, David A

    2009-11-01

    Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to help control the spread of disease. Reverse-transcriptase polymerase chain reaction (RT-PCR) can detect the presence of mRNA (shorter half-life than DNA) with greater potential for detecting viable pathogens. The chromosomally located invA gene required for host invasion by Salmonella is widely used for detection of this pathogen by PCR. Detection of Salmonella was undertaken by real-time RT-PCR (rt-RT-PCR) using newly designed invA gene primers to develop a sensitive and specific assay. Salmonella serovars Typhimurium and Enteritidis were grown (7.68 log(10) CFU/mL) in Luria-Bertani broth overnight at 37 degrees C, and RNA was extracted, followed by rt-RT-PCR with and without SYBR green I and agarose gel electrophoresis. All experiments were replicated at least thrice. Detection for both serovars using traditional RT-PCR was lower ( approximately 10(5) CFU/mL) than rt-RT-PCR (10(3) CFU/mL) by gel electrophoresis. Melt curve analysis showed melt temperatures at 87.5 degrees C with Ct values from 12 to 15 for up to 10(3) CFU/mL and improved to 10(2) CFU/mL after further optimization. Further, addition of RNA internal amplification control constructed using in vitro transcription with a T7 RNA polymerase promoter, to the RT-PCR assay also gave detection limits of 10(2) CFU/mL. Cross-reactivity was not observed against a panel of 21 non-Salmonella bacteria. Heat-inactivated (autoclaved) Salmonella showed faint or no detection by rt-RT-PCR or gel electrophoresis. This method has potential to be applied for the detection of Salmonella serovars in fresh produce and the simultaneous detection of foodborne viral (RNA viruses) and bacterial pathogens in a multiplex format.

  7. Novel approach for detecting prohibited species-specific central nervous system tissue contamination in meat by one-step real-time reverse transcriptase PCR.

    PubMed

    Nagarajan, M M; Longtin, D; Simard, C

    2009-05-01

    The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 10(2) to 10(9) copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.

  8. A quantitative real-time assessment of Buerger exercise on dorsal foot peripheral skin circulation in patients with diabetes foot.

    PubMed

    Chang, Chang-Cheng; Chen, Men-Yen; Shen, Jen-Hsiang; Lin, Yen Bin; Hsu, Wen-Wei; Lin, Bor-Shyh

    2016-11-01

    Buerger exercise can improve the peripheral circulation of lower extremities. However, the evidence and a quantitative assessment of skin perfusion immediately after this exercise in patients with diabetes feet are still rare.We recruited 30 patients with unilateral or bilateral diabetic ulcerated feet in Chang Gung Memorial Hospital, Chia-Yi Branch, from October 2012 to December 2013. Real-time dorsal foot skin perfusion pressures (SPPs) before and after Buerger exercise were measured and analyzed. In addition, the severity of ischemia and the presence of ulcers before exercise were also stratified.A total of 30 patients with a mean age of 63.4 ± 13.7 years old were enrolled in this study. Their mean duration of diabetes was 13.6 ± 8.2 years. Among them, 26 patients had unilateral and 4 patients had bilateral diabetes foot ulcers. Of the 34 wounded feet, 23 (68%) and 9 (27%) feet were classified as Wagner class II and III, respectively. The real-time SPP measurement indicated that Buerger exercise significantly increased the level of SPP by more than 10 mm Hg (n = 46, 58.3 vs 70.0 mm Hg, P < 0.001). In terms of pre-exercise dorsal foot circulation condition, the results showed that Buerger exercise increased the level of SPP in severe ischemia (n = 5, 22.1 vs 37.3 mm Hg, P = 0.043), moderate ischemia (n = 14, 42.2 vs 64.4 mm Hg, P = 0.001), and borderline-normal (n = 7, 52.9 vs 65.4 mm Hg, P = 0.028) groups, respectively. However, the 20 feet with SPP levels more than 60 mm Hg were not improved significantly after exercise (n = 20, 58.3 vs 71.5 mm Hg, P = 0.239). As to the presence of ulcers, Buerger exercise increased the level of SPP in either unwounded feet (n = 12, 58.5 vs 66.0 mm Hg, P = 0.012) or wounded feet (n = 34, 58.3 vs 71.5 mm Hg, P < 0.001). The majority of the ulcers was either completely healed (9/34 = 27%) or still improving (14/34 = 41%).This study quantitatively demonstrates the

  9. A quantitative real-time assessment of Buerger exercise on dorsal foot peripheral skin circulation in patients with diabetes foot

    PubMed Central

    Chang, Chang-Cheng; Chen, Men-Yen; Shen, Jen-Hsiang; Lin, Yen Bin; Hsu, Wen-Wei; Lin, Bor-Shyh

    2016-01-01

    Abstract Buerger exercise can improve the peripheral circulation of lower extremities. However, the evidence and a quantitative assessment of skin perfusion immediately after this exercise in patients with diabetes feet are still rare. We recruited 30 patients with unilateral or bilateral diabetic ulcerated feet in Chang Gung Memorial Hospital, Chia-Yi Branch, from October 2012 to December 2013. Real-time dorsal foot skin perfusion pressures (SPPs) before and after Buerger exercise were measured and analyzed. In addition, the severity of ischemia and the presence of ulcers before exercise were also stratified. A total of 30 patients with a mean age of 63.4 ± 13.7 years old were enrolled in this study. Their mean duration of diabetes was 13.6 ± 8.2 years. Among them, 26 patients had unilateral and 4 patients had bilateral diabetes foot ulcers. Of the 34 wounded feet, 23 (68%) and 9 (27%) feet were classified as Wagner class II and III, respectively. The real-time SPP measurement indicated that Buerger exercise significantly increased the level of SPP by more than 10 mm Hg (n = 46, 58.3 vs 70.0 mm Hg, P < 0.001). In terms of pre-exercise dorsal foot circulation condition, the results showed that Buerger exercise increased the level of SPP in severe ischemia (n = 5, 22.1 vs 37.3 mm Hg, P = 0.043), moderate ischemia (n = 14, 42.2 vs 64.4 mm Hg, P = 0.001), and borderline–normal (n = 7, 52.9 vs 65.4 mm Hg, P = 0.028) groups, respectively. However, the 20 feet with SPP levels more than 60 mm Hg were not improved significantly after exercise (n = 20, 58.3 vs 71.5 mm Hg, P = 0.239). As to the presence of ulcers, Buerger exercise increased the level of SPP in either unwounded feet (n = 12, 58.5 vs 66.0 mm Hg, P = 0.012) or wounded feet (n = 34, 58.3 vs 71.5 mm Hg, P < 0.001). The majority of the ulcers was either completely healed (9/34 = 27%) or still improving (14/34 = 41%). This study quantitatively

  10. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

    PubMed

    Maheshwari, Yogita; Selvaraj, Vijayanandraj; Hajeri, Subhas; Yokomi, Raymond

    2017-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

  11. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR

    PubMed Central

    Hajeri, Subhas

    2017-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR. PMID:28910375

  12. Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

    PubMed

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-06-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests.

  13. Rapid Genome Detection of Schmallenberg Virus and Bovine Viral Diarrhea Virus by Use of Isothermal Amplification Methods and High-Speed Real-Time Reverse Transcriptase PCR

    PubMed Central

    Aebischer, Andrea; Wernike, Kerstin; Beer, Martin

    2014-01-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. PMID:24648561

  14. Porcine reproductive and respiratory syndrome virus: interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods.

    PubMed

    Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte; Errington, Jane; LeBlanc, Neil; Revilla-Fernández, Sandra; Hjulsager, Charlotte; Isaksson, Mats; Stadejek, Tomasz; Beer, Martin; Hoffmann, Bernd

    2012-09-01

    To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

  15. Expression Profiling of the Whole Arabidopsis Shaggy-Like Kinase Multigene Family by Real-Time Reverse Transcriptase-Polymerase Chain Reaction1

    PubMed Central

    Charrier, Bénédicte; Champion, Anthony; Henry, Yves; Kreis, Martin

    2002-01-01

    The recent publication of the complete sequence of the Arabidopsis genome allowed us to identify and characterize the last two members of the SHAGGY-like kinase (AtSK) gene family. As a result, the study of the overall spatio-temporal organization of the whole AtSK family in Arabidopsis has become an achievable and necessary aim to understand the role of each SHAGGY-like kinase during plant development. An analysis of the transcript level of the 10 members of the family has been performed using the technique of real-time quantitative reverse transcriptase-polymerase chain reaction. Transcript levels in several organs, under different growth conditions, were analyzed. To calibrate the results obtained, a number of other genes, such as those coding for the two MAP3Kεs and the two MAP4Kαs, as well as the stress response marker RD29A; the small subunit of the Rubisco photosynthetic enzyme Ats1A; the MEDEA chromatin remodeling factor; and the SCARECROW, ASYMMETRIC LEAVES 1, and SUPERMAN transcription factors all involved in key steps of plant development were used. The analysis of our data revealed that eight of the 10 genes of the AtSK family displayed a pseudo-constitutive expression pattern at the organ level. Conversely, AtSK13 responded to osmotic changes and saline treatment, whereas AtSK31 was flower specific and responded to osmotic changes and darkness. PMID:12376626

  16. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

    NASA Astrophysics Data System (ADS)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-11-01

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

  17. A method for correcting standard-based real-time PCR DNA quantitation when the standard's polymerase reaction efficiency is significantly different from that of the unknown's

    USDA-ARS?s Scientific Manuscript database

    Standard-based real-time, or quantitative, polymerase chain reaction (qPCR) quantitation of an unknown sample’s DNA concentration (i.e., [DNA]-unk) assumes that the concentration dependence of the standard and unknown reactions (related to reaction efficiency, E) are equivalent. In our work with ba...

  18. Quantitation of human papillomavirus type 16 E6 oncogene sequences by real-time or quantitative PCR with EvaGreen.

    PubMed

    Hernández-Arteaga, Socorro; López-Revilla, Rubén

    2008-09-01

    Quantitation of E6 oncogene sequences of the human papillomavirus type 16 by real-time or quantitative PCR (qPCR) is used to determine the viral load, which correlates with the degree of the cervical neoplastic lesions. In the presence of EvaGreen, a new DNA intercalating fluorochrome, we obtained consistent and reproducible qPCR amplification curves and thermal denaturation profiles identical to those of the authentic E6-HPV16 (human papillomavirus 16) genome from the amplification products derived from a construct carrying the E6-HPV16 oncogene. E6-HPV16 quantitation in the presence of EvaGreen, therefore, is reproducible and specific and may be used to determine HPV16 viral load.

  19. Rapid detection method for hepatitis A virus from lettuce by a combination of filtration and integrated cell culture-real-time reverse transcription PCR.

    PubMed

    Hyeon, Ji-Yeon; Chon, Jung-Whan; Park, Chankyu; Lee, Joong-Bok; Choi, In-Soo; Kim, Moo-Sang; Seo, Kun-Ho

    2011-10-01

    We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture-real-time reverse transcription PCR (ICC-real-time RT-PCR) to detect infectious HAV. The most suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC-real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC-real-time RT-PCR detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC-real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC-real-time RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC-real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV in vegetables.

  20. A quantitative real-time PCR method using an X-linked gene for sex typing in pigs.

    PubMed

    Ballester, Maria; Castelló, Anna; Ramayo-Caldas, Yuliaxis; Folch, Josep M

    2013-06-01

    At present, a wide range of molecular sex-typing protocols in wild and domestic animals are available. In pigs, most of these methods are based on PCR amplification of X-Y homologous genes followed by gel electrophoresis which is time-consuming and in some cases expensive. In this paper, we describe, for the first time, a SYBR green-based quantitative real-time PCR (qPCR) assay using an X-linked gene, the glycoprotein M6B, for genetic sexing of pigs. Taking into account the differences in the glycoprotein M6B gene copy number between genders, we determine the correct sex of 54 pig samples from either diaphragm or hair follicle from different breeds using the 2(-ΔΔCT) method for relative quantification. Our qPCR assay represents a quick, inexpensive, and reliable tool for sex determination in pigs. This new protocol could be easily adapted to other species in which the sex determination was required.

  1. A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis

    PubMed Central

    Han, Jae-Ik; Chang, Dong-Woo

    2015-01-01

    Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 µL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes. PMID:26040611

  2. Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert.

    PubMed

    Collonnier, Cécile; Schattner, Alexandra; Berthier, Georges; Boyer, Francine; Coué-Philippe, Géraldine; Diolez, Annick; Duplan, Marie-Noëlle; Fernandez, Sophie; Kebdani, Naïma; Kobilinsky, André; Romaniuk, Marcel; de Beuckeleer, Marc; de Loose, Marc; Windels, Pieter; Bertheau, Yves

    2005-01-01

    T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5' junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5' end of the integrated DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman chemistry.

  3. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve

    PubMed Central

    Rueda-Martínez, Carmen; Fernández, M. Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180–240 days old) and 56 old (300–440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta. PMID:27711171

  4. Universal real-time PCR assay for quantitation and size evaluation of residual cell DNA in human viral vaccines.

    PubMed

    André, Murielle; Reghin, Sylviane; Boussard, Estelle; Lempereur, Laurent; Maisonneuve, Stéphane

    2016-05-01

    Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines.

  5. Evaluation of Quantitative Real-Time PCR as a Hepatitis C Virus Supplementary Test After RIBA Discontinuation.

    PubMed

    Gong, Shunyou; Schmotzer, Christine L; Zhou, Lan

    2016-09-01

    Laboratory testing plays a major role in hepatitis C virus (HCV) diagnosis and patient follow-up. The high false positive rates of HCV screening tests require confirmation through a supplementary test. According to the 2003 CDC guidelines, recombinant immunoblot assay (RIBA) is indispensible to confirm positive screening results and differentiate biologic false positivity from true HCV exposure. However, RIBA has been permanently discontinued since 2011. In the 2013 update of its guidelines, CDC called for further studies on HCV laboratory testing without RIBA. In this study, we analyzed the applicability of quantitative real-time PCR (qPCR) as a supplementary HCV diagnostic test. By comparing our HCV testing performances before and after RIBA discontinuation, we found that omitting RIBA has no significant effect on the accurate and efficient identification of HCV infection, provided that HCV antibody signal-to-cutoff ratio is considered. Furthermore, we proposed a new HCV testing algorithm that incorporates semiquantitative assessment of HCV antibody positivity and HCV viral load measurement by qPCR. By following the algorithm, we were able to address confirmation of positive HCV screening results and to provide useful information generally required by clinicians, including the needs of further laboratory testing or clinical follow-up, as well as HCV viral titers.

  6. Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions.

    PubMed

    Nakayama, T J; Rodrigues, F A; Neumaier, N; Marcelino-Guimarães, F C; Farias, J R B; de Oliveira, M C N; Borém, A; de Oliveira, A C B; Emygdio, B M; Nepomuceno, A L

    2014-02-13

    Quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O2-depleted environments, such as flooding-stressed plants.

  7. Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy.

    PubMed

    Einen, Jørn; Thorseth, Ingunn H; Ovreås, Lise

    2008-05-01

    A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.

  8. Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

    PubMed

    Lin, Pengfei; Lan, Xiangli; Chen, Fenglei; Yang, Yanzhou; Jin, Yaping; Wang, Aihua

    2013-01-01

    The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS), NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

  9. Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

    PubMed Central

    Cunha, Pricila da Silva; Pena, Heloisa B.; D'Angelo, Carla Sustek; Koiffmann, Celia P.; Rosenfeld, Jill A.; Shaffer, Lisa G.; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs. PMID:24839341

  10. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples.

    PubMed

    Green, Hyatt C; Haugland, Richard A; Varma, Manju; Millen, Hana T; Borchardt, Mark A; Field, Katharine G; Walters, William A; Knight, R; Sivaganesan, Mano; Kelty, Catherine A; Shanks, Orin C

    2014-05-01

    Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

  11. Application of quantitative real-time PCR for enumeration of total bacterial, archaeal, and yeast populations in kimchi.

    PubMed

    Park, Eun-Jin; Chang, Ho-Won; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Bae, Jin-Woo

    2009-12-01

    Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45 x 10(6) which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.

  12. Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods.

    PubMed

    Klanicova, B; Slana, I; Roubal, P; Pavlik, I; Kralik, P

    2012-07-02

    Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

  13. Analyzing the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative Real-Time Polymerase Chain Reaction.

    PubMed

    Denton, Donna; Kumar, Sharad

    2015-07-01

    A useful complement to animal studies is the use of Drosophila cell lines to analyze cell-death responses. There are numerous Drosophila cell lines available, such as S2 cells, which possess the advantages of being semi-adherent, fast growing, relatively robust, and useful for transfection and knockdown studies, whereas other lines, such as mbn2, are more suitable for analyzing hormone-induced cell death and gene expression. Drosophila cell lines are very amenable to knockdown studies as the cells take up double-stranded RNA (dsRNA) from the medium, initiating gene silencing and resulting in a high level of gene knockdown. This means that the cell lines are useful for investigating the response to death stimuli, following gene knockdown, by examining the expression of cell-death genes. This protocol describes the synthesis of dsRNA for treatment of Drosophila cells and the subsequent analysis of cell-death gene expression by quantitative real-time polymerase chain reaction (qPCR). © 2015 Cold Spring Harbor Laboratory Press.

  14. Application of real-time quantitative PCR to molecular analysis of Candida albicans strains exhibiting reduced susceptibility to azoles.

    PubMed

    Chau, Andrew S; Mendrick, Cara A; Sabatelli, Frank J; Loebenberg, David; McNicholas, Paul M

    2004-06-01

    Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1, in a collection of 14 fluconazole-susceptible Candida albicans isolates. For each gene, average expression levels and variations within the population were determined. These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis. For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above. Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates. In contrast, MDR1, ERG11, CDR1, and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles. In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11. Select ERG11 alleles were expressed in Saccharomyces cerevisiae; all of the alleles tested conferred reduced susceptibility to fluconazole. Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility.

  15. Identification of suitable reference genes for quantitative real-time PCR normalization in blotched snakehead Channa maculata.

    PubMed

    Mao, H; Chen, K; Zhu, X; Luo, Q; Zhao, J; Li, W; Wu, X; Xu, H

    2017-04-07

    A systematic study was conducted to identify reliable reference genes for normalization of gene expression analysis in the blotched snakehead Channa maculata under normal physiological conditions. Firstly, the partial complementary (c)DNA of nine candidate reference genes (actb, tmem104, ube2l3, ef1α, churc1, tmem256, rpl13a, sep15 and g6pd) were cloned from C. maculata. The expression levels of these genes were then assessed in embryos of different developmental stages and various tissue types of adult fish using quantitative real-time (qrt-)PCR. RefFinder algorithm was used to evaluate the expression stability of these genes based on their cycle-threshold (Ct ) values in the qrt-PCR analysis. Results showed that there was no single best reference gene for all stages of embryos and adult tissues tested. Furthermore, it was found that, among the nine candidate genes tested, actb and tmem104 were the most stable reference genes across adult tissue types, while sep15 and tmem256 were the most stable ones across developmental stages of embryos. These stable reference genes are recommended for normalization of gene expression analysis in C. maculata.

  16. Quantitative analysis of herpes virus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR

    USGS Publications Warehouse

    Quackenbush, S.L.; Casey, R.N.; Murcek, R.J.; Paul, T.A.; Work, T.M.; Limpus, C.J.; Chaves, A.; duToit, L.; Perez, J.V.; Aguirre, A.A.; Spraker, T.R.; Horrocks, J.A.; Vermeer, L.A.; Balazs, G.S.; Casey, J.W.

    2001-01-01

    Quantitative real-time PCR has been used to measure fibropapilloma-associated turtle herpesvirus (FPTHV) pol DNA loads in fibropapillomas, fibromas, and uninvolved tissues of green, loggerhead, and olive ridley turtles from Hawaii, Florida, Costa Rica, Australia, Mexico, and the West Indies. The viral DNA loads from tumors obtained from terminal animals were relatively homogenous (range 2a??20 copies/cell), whereas DNA copy numbers from biopsied tumors and skin of otherwise healthy turtles displayed a wide variation (range 0.001a??170 copies/cell) and may reflect the stage of tumor development. FPTHV DNA loads in tumors were 2.5a??4.5 logs higher than in uninvolved skin from the same animal regardless of geographic location, further implying a role for FPTHV in the etiology of fibropapillomatosis. Although FPTHV pol sequences amplified from tumors are highly related to each other, single signature amino acid substitutions distinguish the Australia/Hawaii, Mexico/Costa Rica, and Florida/Caribbean groups.

  17. Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.

    PubMed

    Mamidala, Praveen; Rajarapu, Swapna P; Jones, Susan C; Mittapalli, Omprakash

    2011-07-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

  18. DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

    PubMed Central

    Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael

    2016-01-01

    Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. PMID:28952569

  19. [Detection of Plasmodium falciparum by using magnetic nanoparticles separation-based quantitative real-time PCR assay].

    PubMed

    Wang, Fei; Tian, Yin; Yang, Jing; Sun, Fu-Jun; Sun, Ning; Liu, Bi-Yong; Tian, Rui; Ge, Guang-Lu; Zou, Ming-qiang; Deng, Cong-liang; Liu, Yi

    2014-10-01

    To establish a magnetic nanoparticles separation-based quantitative real-time PCR (RT-PCR) assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and prevention of imported malaria. According to the conserved sequences of the P. falciparum genome 18SrRNA, the species-specific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard, fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluated. The relationship between the threshold cycle (Ct) and logarithm of initial templates copies was linear over a range of 2.5 x 10(1) to 2.5 x 10(8) copies/μl (R2 = 0.999). Among 13 subjects of entry frontier, a P. falciparum carrier with low load was detected by using the assay and none was detected with the conventional examinations (microscopic examinations and rapid tests). This assay shows a high sensitivity in detection of P. falciparum, with rapid and accurate characteristics, and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.

  20. Effectiveness of Quantitative Real Time PCR in Long-Term Follow-up of Chronic Myeloid Leukemia Patients.

    PubMed

    Savasoglu, Kaan; Payzin, Kadriye Bahriye; Ozdemirkiran, Fusun; Berber, Belgin

    2015-08-01

    To determine the use of the Quantitative Real Time PCR (RQ-PCR) assay follow-up with Chronic Myeloid Leukemia (CML) patients. Cross-sectional observational. Izmir Ataturk Education and Research Hospital, Izmir, Turkey, from 2009 to 2013. Cytogenetic, FISH, RQ-PCR test results from 177 CMLpatients' materials selected between 2009 - 2013 years was set up for comparison analysis. Statistical analysis was performed to compare between FISH, karyotype and RQ-PCR results of the patients. Karyotyping and FISH specificity and sensitivity rates determined by ROC analysis compared with RQ-PCR results. Chi-square test was used to compare test failure rates. Sensitivity and specificity values were determined for karyotyping 17.6 - 98% (p=0.118, p > 0.05) and for FISH 22.5 - 96% (p=0.064, p > 0.05) respectively. FISH sensitivity was slightly higher than karyotyping but there was calculated a strong correlation between them (p < 0.001). RQ-PCR test failure rate did not correlate with other two tests (p > 0.05); however, karyotyping and FISH test failure rate was statistically significant (p < 0.001). Besides, the situation needed for karyotype analysis, RQ-PCR assay can be used alone in the follow-up of CMLdisease.

  1. Detection and quantitation of HTLV-1 and HTLV-2 mRNA species by real-time RT-PCR.

    PubMed

    Li, Min; Green, Patrick L

    2007-06-01

    HTLV-1 and HTLV-2 are highly related delta-retroviruses that infect and transform T-lymphocytes, but have distinct pathogenic properties. HTLV replication and survival requires the expression of multiple gene products from an unspliced and a series of highly related alternatively spliced mRNA species. To date, the comparative levels of all known HTLV-1 and HTLV-2 viral mRNAs in different transformed cell lines and at different stages of virus infection have not been assessed. In this study, we compiled a series of oligonucleotide primer pairs and probes to quantify both HTLV-1 and HTLV-2 mRNA species using real-time RT-PCR. The optimized reaction for detection of each mRNA had amplification efficiency greater than 90% with a linear range spanning 25-2.5 x 10(7) copies. The R(2)'s of all standard curves were greater than 0.97. Quantitation of HTLV mRNAs between different cell lines showed variability (gag/pol>or=tax/rex>env>or=accessory proteins), but the overall levels of each mRNA relative to each other within a cell line were similar. These results provide a method to quantify all specific mRNAs from both HTLV-1 and HTLV-2, which can be used to evaluate further viral gene expression and correlate transcript levels to key stages of the virus life cycle and ultimately, pathogenesis.

  2. Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

    PubMed

    Luan, Huaibiao; Wang, Yixin; Li, Yang; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2016-09-01

    Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers). © 2016 Poultry Science Association Inc.

  3. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

    PubMed

    Rueda-Martínez, Carmen; Fernández, M Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

  4. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    PubMed

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  5. Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

    PubMed

    Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

    2017-07-01

    Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  6. Selection of suitable reference genes for quantitative real-time PCR in apoptosis-induced MCF-7 breast cancer cells.

    PubMed

    Ferreira, Eloise; Cronjé, Marianne J

    2012-02-01

    Apoptosis is induced in MCF-7 breast cancer cells following treatment with salicylic acid (20 mM), either in the presence or absence of a heat shock (42°C for 30 min). In order to study the alterations of apoptotic genes with quantitative real-time PCR (qPCR), suitable genes with unchanged expression following the treatments is required for normalizing the gene expression levels. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), Histone H2A (HIST), constitutively expressed heat shock protein 70 (HSC70) and tyrosine 3-monooxygenase/trytophan 5 monooxygenase activation protein, 14-3-3 (YWHAZ) were evaluated as appropriate reference genes. Analysis of gene expression data with one-way ANOVA, geNorm and NormFinder identified HIST and YWHAZ as the least affected during the induction of apoptosis by the different treatments, and is the most suitable gene-pair for normalization during qPCR analysis in MCF-7 breast cancer cells undergoing apoptosis following treatment with SA and/or HS.

  7. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti.

    PubMed

    Dzaki, Najat; Ramli, Karima N; Azlan, Azali; Ishak, Intan H; Azzam, Ghows

    2017-03-16

    The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.

  8. Accurate, fast and cost-effective diagnostic test for monosomy 1p36 using real-time quantitative PCR.

    PubMed

    Cunha, Pricila da Silva; Pena, Heloisa B; D'Angelo, Carla Sustek; Koiffmann, Celia P; Rosenfeld, Jill A; Shaffer, Lisa G; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5-0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

  9. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti

    PubMed Central

    Dzaki, Najat; Ramli, Karima N.; Azlan, Azali; Ishak, Intan H.; Azzam, Ghows

    2017-01-01

    The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research. PMID:28300076

  10. [Detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues by real-time fluorescent quantitative polymerase chain reaction].

    PubMed

    Ye, Feng; Chen, Yu; He, Du; Jian, Shu-yu; Zheng, Ke; Li, Gan-di; Bu, Hong

    2013-08-01

    To investigate the feasibility of real-time fluorescent quantitative (qPCR) assay in detecting mycobacterium tuberculosis complex (MTB) in paraffin embedded tissues for diagnostic purpose. Using qPCR assay, 1000 consecutive formalin-fixed and paraffin embedded (FFPE) tissues (from 2011 to 2012) suspected of MTB infection were tested by amplifying the MTB specific insertion sequence 6110 (IS6110). The specificity of the PCR product was confirmed by Sanger sequencing as compared with the MTB genomic DNA of the IS6110 sequence. Tissues with Ziehl-Neelsen acid-fast staining were used as control. In the 1000 samples, 513 were positive for mycobacterium by Ziehl-Neelsen acid-fast staining (detection rate 51.3%); whereas 546 were MTB positive by qPCR assay (detection rate 54.6%). Concordance rate for both assays was 73.1%. The diagnosis rate increased by 14.4% by combinination of Ziehl-Neelsen acid-fast staining and qPCR results. More interestingly, by analyzing the Ziehl-Neelsen acid-fast staining and qPCR results three cases of M.leprae infection and four cases of non-tuberculous Mycobacterium (NTM) infection were identified. qPCR detection of MTB in FFPE tissue is more sensitive than Ziehl-Neelsen acid-fast staining assay. Combination of these two assays can increase the detection rate and also identify some rare cases of NTM infection.

  11. Quantitative imaging reveals real-time Pou5f3–Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish

    PubMed Central

    Perez-Camps, Mireia; Tian, Jing; Chng, Serene C; Sem, Kai Pin; Sudhaharan, Thankiah; Teh, Cathleen; Wachsmuth, Malte; Korzh, Vladimir; Ahmed, Sohail; Reversade, Bruno

    2016-01-01

    Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3–Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3–Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3–Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis. DOI: http://dx.doi.org/10.7554/eLife.11475.001 PMID:27684073

  12. A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis.

    PubMed

    Han, Jae-Ik; Chang, Dong-Woo; Na, Ki-Jeong

    2015-01-01

    Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 mL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes.

  13. Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba.

    PubMed

    Lin, Liyuan; Han, Xiaojiao; Chen, Yicun; Wu, Qingke; Wang, Yangdong

    2013-12-01

    Quantitative real-time PCR has emerged as a highly sensitive and widely used method for detection of gene expression profiles, via which accurate detection depends on reliable normalization. Since no single control is appropriate for all experimental treatments, it is generally advocated to select suitable internal controls prior