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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. Analysis of Liver Connexin Expression Using Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction.

    PubMed

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

    2016-01-01

    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 RNA 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

  3. Real-Time Reverse Transcription-PCR Quantitation of Substance P Receptor (NK-1R) mRNA

    PubMed Central

    Lai, Jian-Ping; Douglas, Steven D.; Wang, Yan-Jian; Ho, Wen-Zhe

    2005-01-01

    The substance P (SP)-preferring receptor, neurokinin-1 receptor (NK-1R), has an important role in inflammation, immune regulation, and viral infection. We applied a newly developed real-time reverse transcription (RT)-PCR assay to quantify NK-1R mRNA in human neuronal cell line (NT-2N), a human B-cell line (IM9), monocyte-derived macrophages (MDM), peripheral blood lymphocytes (PBL), and human astroglioma cells (U87 MG). The NK-1R real-time RT-PCR assay has a sensitivity of 100 mRNA copies, with a dynamic range of detection between 102 and 107 copies of NK-1R gene transcripts per reaction. This assay is highly reproducible, with an intraassay coefficient variation of threshold cycle (Ct) of less than 1.9%. The NK-1R real-time RT-PCR is highly sensitive for quantitative determination of NK-1R mRNA in human immune cells (MDM and PBL) that express low levels of NK-1R mRNA. In addition, the assay has the ability to accurately quantitate the dynamic changes in NK-1R mRNA expression in interleukin-1β-stimulated U87 MG. These data indicate that the NK-1R real-time RT-PCR has potential for a wide application in investigation of NK-1R expression at the mRNA level under physiological and pathological conditions in both the central nervous system and the immune system. PMID:15817763

  4. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  6. Rapid detection of enteroviruses in small volumes of natural waters by real-time quantitative reverse transcriptase PCR.

    PubMed

    Fuhrman, Jed A; Liang, Xiaolin; Noble, Rachel T

    2005-08-01

    Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r2= 0.99) in fresh water and 23% (r2= 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <10(4) enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%, r2= 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%; r2= 0.93; HA 12%, r2= 0.87). The optimized method was used with 1-liter field samples from two very different freshwater "creeks" that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples. PMID:16085845

  7. 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. PMID:26912233

  8. 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. PMID:27246908

  9. 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. PMID:26843055

  10. Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

    PubMed Central

    Pizeta Semighini, Camile; Marins, Mozart; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2002-01-01

    The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background. PMID:11872487

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Real-time quantitative reverse transcription-polymerase chain reaction to detect propionibacterial ribosomal RNA in the lymph nodes of Chinese patients with sarcoidosis.

    PubMed

    Zhou, Y; Wei, Y-R; Zhang, Y; Du, S-S; Baughman, R P; Li, H-P

    2015-09-01

    The aim of this study was to investigate the diagnostic value of using the copy number of propionibacterial rRNA as a biomarker for sarcoidosis. Ribosomal RNA of Propionibacterium acnes and P. granulosum was measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using formalin-fixed and paraffin-embedded tissue of lymph node biopsy from 65 Chinese patients with sarcoidosis, 45 with tuberculosis and 50 controls with other diseases (23 with non-specific lymphadenitis and 27 with mediastinal lymph node metastasis from lung cancer). The receiver operating characteristic (ROC) curve was analysed to determine an optimal cut-off value for diagnosis, and the diagnostic accuracy of the cut-off value was evaluated in additional tissue samples [24 patients with sarcoidosis and 22 with tuberculosis (TB)]. P. acnes or P. granulosum rRNA was detected in 48 of the 65 sarcoidosis samples but only in four of the 45 TB samples and three of the 50 control samples. Analysis of the ROC curve revealed that an optimal cut-off value of the copy number of propionibacterial rRNA for diagnosis of sarcoidosis was 50·5 copies/ml with a sensitivity and specificity of 73·8 and 92·6%, respectively. Based on the cut-off value, 19 of the 24 additional sarcoidosis samples exhibited positive P. acnes or P. granulosum, whereas only one of the 22 additional TB samples was positive, resulting in a sensitivity and specificity of 79·2 and 95·5%, respectively. These findings suggest that propionibacteria might be associated with sarcoidosis granulomatous inflammation. Detection of propionibacterial rRNA by RT-PCR might possibly distinguish sarcoidosis from TB. PMID:25959360

  14. Real-time quantitative reverse transcription–polymerase chain reaction to detect propionibacterial ribosomal RNA in the lymph nodes of Chinese patients with sarcoidosis

    PubMed Central

    Zhou, Y; Wei, Y-R; Zhang, Y; Du, S-S; Baughman, R P; Li, H-P

    2015-01-01

    The aim of this study was to investigate the diagnostic value of using the copy number of propionibacterial rRNA as a biomarker for sarcoidosis. Ribosomal RNA of Propionibacterium acnes and P. granulosum was measured by real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) using formalin-fixed and paraffin-embedded tissue of lymph node biopsy from 65 Chinese patients with sarcoidosis, 45 with tuberculosis and 50 controls with other diseases (23 with non-specific lymphadenitis and 27 with mediastinal lymph node metastasis from lung cancer). The receiver operating characteristic (ROC) curve was analysed to determine an optimal cut-off value for diagnosis, and the diagnostic accuracy of the cut-off value was evaluated in additional tissue samples [24 patients with sarcoidosis and 22 with tuberculosis (TB)]. P. acnes or P. granulosum rRNA was detected in 48 of the 65 sarcoidosis samples but only in four of the 45 TB samples and three of the 50 control samples. Analysis of the ROC curve revealed that an optimal cut-off value of the copy number of propionibacterial rRNA for diagnosis of sarcoidosis was 50·5 copies/ml with a sensitivity and specificity of 73·8 and 92·6%, respectively. Based on the cut-off value, 19 of the 24 additional sarcoidosis samples exhibited positive P. acnes or P. granulosum, whereas only one of the 22 additional TB samples was positive, resulting in a sensitivity and specificity of 79·2 and 95·5%, respectively. These findings suggest that propionibacteria might be associated with sarcoidosis granulomatous inflammation. Detection of propionibacterial rRNA by RT–PCR might possibly distinguish sarcoidosis from TB. PMID:25959360

  15. Application of quantitative real-time reverse transcription-PCR in assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum in vitro.

    PubMed

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

    2005-11-01

    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 (C(T)([P18S])) and host cells (C(T)([H18S])), and evaluates the relative expression between parasite and host rRNA levels (i.e., deltaC(T) = C(T)([P18S]) - C(T)([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 (deltadeltaC(T)) 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 approximately 3 h) in dead parasites is faster than that of DNA (99% in approximately 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 micro/ml and 89.7 to 119.0 microg/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

  16. 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

  17. New In Situ Capture Quantitative (Real-Time) Reverse Transcription-PCR Method as an Alternative Approach for Determining Inactivation of Tulane Virus

    PubMed Central

    Wang, Dapeng; Xu, Shuxia; Yang, David; Young, Glenn M.

    2014-01-01

    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

  18. CD44 variant exon 6 expressions in colon cancer assessed by quantitative analysis using real time reverse transcriptase-polymerase chain reaction.

    PubMed

    Yamada, Yoichi; Itano, Naoki; Narimatsu, Hisashi; Kudo, Takashi; Hirohashi, Setsuo; Ochiai, Atsushi; Tohnai, Iwai; Ueda, Minoru; Kimata, Koji

    2003-01-01

    CD44 is a family of transmembrane glycoproteins that serve as a major receptor for hyaluronate and the splice variants play a very important role in tumor progression and metastasis. We examined the relationship between cancer progression and mRNA levels of CD44 variant exon 6 (CD44v6) in specimens of colon cancer at different diagnostic stages from 31 patients using real time RT-PCR analysis. Increased mRNA levels of CD44v6 were observed in 82% of the specimens in comparison with those in the corresponding non-cancerous tissue specimens. A statistically significant correlation between the CD44v6 expression and the cancerous state was found in most specimens at all Dukes stages. None of the other parameters were related to the expression in the cancerous specimens. Quantitative real time RT-PCR analysis showed that there was no correlation of CD44v6 expression with tumor progression, although CD44v6 is upregulated in transformation. Thus, CD44v6 expression may be a clinically useful indicator of colon cancer. PMID:14534719

  19. Quantitative Detection of Citrus tristeza virus (CTV) in Citrus and Aphids by Real-time Reverse Transcription-PCR (TaqMan®)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Routine detection of Citrus tristeza virus (CTV) is by enzyme-linked immunosorbent (ELISA) and direct tissue blot immunoassays. Reverse transcription (RT) polymerase chain reaction (PCR) has also been developed for CTV detection which is more sensitive than serology. We developed a quantitative an...

  20. 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). PMID:25486080

  1. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    PubMed

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, p<0.001). hTR levels however, were high in all tumors and independently of the presence of germ cells also in the benign tissue control group. hTERT mRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype. PMID:12168080

  2. 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. PMID:19376910

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

    PubMed

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

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Quantitative Detection of Spiroplasma Citri by Real Time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop an accurate and rapid method to detect Spiroplasma citri, the causal agent of citrus stubborn disease for use in epidemiology studies. Quantitative real-time PCR was developed for detection of S. citri. Two sets of primers based on sequences from the P58 putative adhesin ...

  6. Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.

    PubMed

    Lizée, Gregory; Aerts, Joeri L; Gonzales, Monica I; Chinnasamy, Nachimuthu; Morgan, Richard A; Topalian, Suzanne L

    2003-04-10

    The use of lentiviral vectors for basic research and potential future clinical applications requires methodologies that can accurately determine lentiviral titers and monitor viral transgene expression within target cells, beyond the context of reporter genes typically used for this purpose. Here we describe a quantitative RT-PCR (qRT-PCR) method that achieves both goals using primer sequences that are specific for the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), an enhancer contained in many retroviral vectors and that is incorporated in the 3' UTR of nascent transgene transcripts. Quantitation of titers of three recombinant lentiviruses, genetically identical except for the transgene, demonstrated consistent differences in titer that were likely due to transgene-associated toxicity in producer cells and target cells. Viruses encoding the tumor-associated antigens tyrosinase and neo-poly(A) polymerase yielded reproducibly lower titers than a virus encoding enhanced green fluorescent protein (GFP) at the viral RNA, integrated DNA, and transgene mRNA levels, as measured by WPRE qPCR. Furthermore, the magnitude of differences in expression of the three transgenes in transduced target cells could not have been predicted by measuring vector DNA integration events. Since transgene expression in target cells is the most common goal of lentiviral transduction, and since methods to quantify transgene expression on the protein level are not always readily available, qRT-PCR based on a nucleotide sequence included in the transcript provides a useful tool for titering novel recombinant lentiviruses. PMID:12718761

  7. 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

  8. Real time inverse filter focusing through iterative time reversal.

    PubMed

    Montaldo, Gabriel; Tanter, Mickaël; Fink, Mathias

    2004-02-01

    In order to achieve an optimal focusing through heterogeneous media we need to build the inverse filter of the propagation operator. Time reversal is an easy and robust way to achieve such an inverse filter in nondissipative media. However, as soon as losses appear in the medium, time reversal is not equivalent to the inverse filter anymore. Consequently, it does not produce the optimal focusing and beam degradations may appear. In such cases, we showed in previous works that the optimal focusing can be recovered by using the so-called spatiotemporal inverse filter technique. This process requires the presence of a complete set of receivers inside the medium. It allows one to reach the optimal focusing even in extreme situations such as ultrasonic focusing through human skull or audible sound focusing in strongly reverberant rooms. But, this technique is time consuming and implied fastidious numerical calculations. In this paper we propose a new way to process this inverse filter focusing technique in real time and without any calculation. The new process is based on iterative time reversal process. Contrary to the classical inverse filter technique, this iteration does not require any computation and achieves the inverse filter in an experimental way using wave propagation instead of computational power. The convergence from time reversal to inverse filter during the iterative process is theoretically explained. Finally, the feasibility of this iterative technique is experimentally demonstrated for ultrasound applications. PMID:15000188

  9. Real time blood testing using quantitative phase imaging.

    PubMed

    Pham, Hoa V; Bhaduri, Basanta; Tangella, Krishnarao; Best-Popescu, Catherine; Popescu, Gabriel

    2013-01-01

    We demonstrate a real-time blood testing system that can provide remote diagnosis with minimal human intervention in economically challenged areas. Our instrument combines novel advances in label-free optical imaging with parallel computing. Specifically, we use quantitative phase imaging for extracting red blood cell morphology with nanoscale sensitivity and NVIDIA's CUDA programming language to perform real time cellular-level analysis. While the blood smear is translated through focus, our system is able to segment and analyze all the cells in the one megapixel field of view, at a rate of 40 frames/s. The variety of diagnostic parameters measured from each cell (e.g., surface area, sphericity, and minimum cylindrical diameter) are currently not available with current state of the art clinical instruments. In addition, we show that our instrument correctly recovers the red blood cell volume distribution, as evidenced by the excellent agreement with the cell counter results obtained on normal patients and those with microcytic and macrocytic anemia. The final data outputted by our instrument represent arrays of numbers associated with these morphological parameters and not images. Thus, the memory necessary to store these data is of the order of kilobytes, which allows for their remote transmission via, for example, the cellular network. We envision that such a system will dramatically increase access for blood testing and furthermore, may pave the way to digital hematology. PMID:23405194

  10. 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

  11. 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.

  12. 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...

  13. 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.

  14. 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.; Liu, Frank W.; Otten, David M.; Stevenson, David K.; Cheong, Wai-Fung; Kermit, Eben L.

    1997-12-01

    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.

  15. Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction.

    PubMed

    Capote, Nieves; Gorris, M Teresa; Martínez, M Carmen; Asensio, Margarita; Olmos, Antonio; Cambra, Mariano

    2006-03-01

    ABSTRACT The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3' minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected. PMID:18944448

  16. 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

  17. 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.

  18. Quantitative Analysis of Copy Number Variants Based on Real-Time LightCycler PCR

    PubMed Central

    Ma, Lijiang; Chung, Wendy K.

    2014-01-01

    Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples. PMID:24510682

  19. Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

    PubMed Central

    Guilbaud, Morgan; de Coppet, Pierre; Bourion, Fabrice; Rachman, Cinta; Prévost, Hervé; Dousset, Xavier

    2005-01-01

    A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 102 CFU/cm2. PMID:15812058

  20. 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

  1. Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.

    PubMed

    Lewis, April M; Rice, Kelly C

    2016-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression. PMID:25646613

  2. 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.

  3. 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. PMID:24395270

  4. New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

    PubMed Central

    Gueimonde, Miguel; Tölkkö, Satu; Korpimäki, Teemu; Salminen, Seppo

    2004-01-01

    The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 104 cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces. PMID:15240297

  5. Reference Gene Selection for Quantitative Real-time PCR Normalization in Quercus suber

    PubMed Central

    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. PMID:22529976

  6. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp.

    PubMed

    Mekata, Tohru; Sudhakaran, Raja; Kono, Tomoya; U-taynapun, Kittichon; Supamattaya, Kidchakan; Suzuki, Yoshihiro; Sakai, Masahiro; Itami, Toshiaki

    2009-12-01

    A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 degrees C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1alpha, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture. PMID:19646483

  7. Type-A influenza virus detection and quantitation by real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) is a relatively new technology which has been used for AIV detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of RRT-PCR are: quantitative nature, scalability, cost, high sensitivity, high specificity, and ...

  8. 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...

  9. DETECTION AND QUANTITATION OF SOLENOPSIS INVICTA VIRUS IN FIRE ANTS BY REAL-TIME PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of Solenopsis invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) f...

  10. Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay.

    PubMed

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-05-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  11. Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay

    PubMed Central

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-01-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. 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...

  14. 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. PMID:16318892

  15. Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

    PubMed Central

    Emery, Shannon L.; Bowen, Michael D.; Newton, Bruce R.; Winchell, Jonas M.; Meyer, Richard F.; Tong, Suxiang; Cook, Byron T.; Holloway, Brian P.; McCaustland, Karen A.; Rota, Paul A.; Bankamp, Bettina; Lowe, Luis E.; Ksiazek, Tom G.; Bellini, William J.; Anderson, Larry J.

    2004-01-01

    A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection. PMID:15030703

  16. A new method for robust quantitative and qualitative analysis of real-time PCR

    PubMed Central

    Shain, Eric B.; Clemens, John M.

    2008-01-01

    An automated data analysis method for real-time PCR needs to exhibit robustness to the factors that routinely impact the measurement and analysis of real-time PCR data. Robust analysis is paramount to providing the same interpretation for results regardless of the skill of the operator performing or reviewing the work. We present a new method for analysis of real-time PCR data, the maxRatio method, which identifies a consistent point within or very near the exponential region of the PCR signal without requiring user intervention. Compared to other analytical techniques that generate only a cycle number, maxRatio generates several measurements of amplification including cycle numbers and relative measures of amplification efficiency and curve shape. By using these values, the maxRatio method can make highly reliable reactive/nonreactive determination along with quantitative evaluation. Application of the maxRatio method to the analysis of quantitative and qualitative real-time PCR assays is shown along with examples of method robustness to, and detection of, amplification response anomalies. PMID:18603594

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

    PubMed

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-01

    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. PMID:27371921

  18. Selection of Reference Genes for Quantitative Real-Time PCR in Bamboo (Phyllostachys edulis)

    PubMed Central

    Fan, Chunjie; Ma, Jinmin; Guo, Qirong; Li, Xiaotie; Wang, Hui; Lu, Mengzhu

    2013-01-01

    Background The Moso bamboo (Phyllostachys edulis) is one of the most important forestry resources and plays essential ecological roles in southern China. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression data related to the unique traits of Moso bamboo will undoubtedly follow. Reverse transcription quantitative real-time PCR ((RT-)qPCR) is a widely used method for gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. Result In this study, 14 candidate reference genes were chosen, and their expression levels were assessed by (RT-)qPCR in a set of six tissue samples (root, stem, mature stem, leaf, flower, and leaf sheath) and at two developmental stages (before and after flowering) in bamboo specimens obtained in three locations. The stability and suitability of the candidate reference genes were validated using the geNorm, NormFinder and BestKeeper programs. The results showed that TIP41 and NTB were suitable reference genes across all the tissues and at the different developmental stages examined in this study. While the expression of the NTB, TIP41 and UBQ were the mostly stable in different plant tissues samples, the expression of the TIP41, NTB and CAC were ranked the most stable in bamboo plants at various developmental stages. AP2-like gene was further assessed by using the reference genes TIP41 and NTB in comparison to ACT. Significant difference of the expression profile of AP2-like demonstrated the importance of choosing adequate reference genes in bamboo. Conclusion TIP41 and NTB were found to be homogeneously expressed and were adequate for normalization purposes, showing equivalent transcript levels in different samples. They are therefore the recommended reference genes for measuring gene expression in P. edulis. PMID:23437174

  19. Expression profiling by real-time quantitative polymerase chain reaction (RT-qPCR).

    PubMed

    Lech, Maciej; Anders, Hans-Joachim

    2014-01-01

    Real-time quantitative PCR is a variation of the standard PCR technique that is commonly used to quantify nucleic acid. However, in this technique the amount of amplified specific sequence can be quantified at each stage of the PCR cycle. If investigated sequence is present in large number of copies in particular sample, amplification product is detected already in earlier cycles; if the sequence is rare, amplification is observed in later cycles. Quantification of amplified product is acquired using fluorescent probes or fluorescent DNA-binding dyes. Accumulation of fluorescent signal can be measured by real-time PCR instruments during each of 35-45 cycwwles of the PCR reaction, which simplify the procedure by eliminating the visualization of the amplified products using gel electrophoresis. Real-time-PCR allows quantifying the amount of product already during the PCR reaction as soon as it is detectable. Correctly performed, this method may be used for precise gene expression analysis in life science, medicine, and diagnostics and has become the standard method of choice for the quantification of mRNA. However in the past few years it became obvious that real-time PCR is complex and variability of RNA templates, assay designs, inappropriate data normalization, and data interpretation may cause diverse analytical problems. PMID:24957236

  20. 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

  1. Real-time Assessment of Flow Reversal in an Eccentric Arterial Stenotic Model

    PubMed Central

    Ai, Lisong; Zhang, Lequan; Dai, Wangde; Hu, Changhong; Shung, K. Kirk; Hsiai, Tzung K.

    2010-01-01

    Plaque rupture is the leading cause of acute coronary syndromes and stroke. Plaque formation, or otherwise known as stenosis, preferentially occurs in the regions of arterial bifurcation or curvatures. To date, real-time assessment of stenosis-induced flow reversal remains a clinical challenge. By interfacing Micro-electro-mechanical Systems (MEMS) thermal sensors with the high frequency Pulsed Wave (PW) Doppler ultrasound, we proposed to assess flow reversal in the presence of an eccentric stenosis. We developed a 3-D stenotic model (inner diameter of 6 mm, an eccentric stenosis with a height of 2.75mm and width of 21 mm) simulating a superficial arterial vessel. We demonstrated that heat transfer from the sensing element (2 × 80 μm) to the flow field peaked as a function of flow rates at the throat of the stenosis alone the center/midline of arterial model, and dropped downstream from the stenosis where flow reversal was detected by the high frequency ultrasound device at 45 MHz. Computational fluid dynamics (CFD) codes were in agreement with the ultrasound-acquired flow profiles upstream, downstream, and at the throat of the stenosis. Hence, we characterized regions of eccentric stenosis in terms of changes in heat transfer alone the midline of vessel and identified points of flow reversal with high spatial and temporal resolution. PMID:20655537

  2. Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses▿

    PubMed Central

    Lu, Xiaoyan; Holloway, Brian; Dare, Ryan K.; Kuypers, Jane; Yagi, Shigeo; Williams, John V.; Hall, Caroline B.; Erdman, Dean D.

    2008-01-01

    Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5′ noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 × 105 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRV-culture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEV-positive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff. PMID:18057136

  3. 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. PMID:19602858

  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. PMID:24978968

  5. 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.

  6. 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

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

    PubMed

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

    2016-06-01

    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

  8. 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

  9. Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

    PubMed Central

    Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

    2006-01-01

    A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages. PMID:16957227

  10. 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. PMID:27557577

  11. 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.

  12. 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

  13. Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

    PubMed Central

    Wise, Mark G.; Siragusa, Gregory R.

    2005-01-01

    Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract. PMID:16000804

  14. Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

    PubMed Central

    Wylie, Todd N.; Wylie, Kristine M.; Buller, Richard S.; Cannella, Maria

    2015-01-01

    We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses. PMID:26063859

  15. Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay.

    PubMed

    Wylie, Todd N; Wylie, Kristine M; Buller, Richard S; Cannella, Maria; Storch, Gregory A

    2015-08-01

    We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses. PMID:26063859

  16. 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.

  17. Quantitative analysis of real-time tissue elastography for evaluation of liver fibrosis

    PubMed Central

    Shi, Ying; Wang, Xing-Hua; Zhang, Huan-Hu; Zhang, Hai-Qing; Tu, Ji-Zheng; Wei, Kun; Li, Juan; Liu, Xiao-Li

    2014-01-01

    The present study aimed to investigate the feasibility of quantitative analysis of liver fibrosis using real-time tissue elastography (RTE) and its pathological and molecule biological basis. Methods: Fifty-four New Zealand rabbits were subcutaneously injected with thioacetamide (TAA) to induce liver fibrosis as the model group, and another eight New Zealand rabbits served as the normal control group. Four rabbits were randomly taken every two weeks for real-time tissue elastography (RTE) and quantitative analysis of tissue diffusion. The obtained twelve characteristic quantities included relative mean value (MEAN), standard deviation (SD), blue area % (% AREA), complexity (COMP), kurtosis (KURT), skewness (SKEW), contrast (CONT), entropy (ENT), inverse different moment (IDM), angular secon moment (ASM), correlation (CORR) and liver fibrosis index (LF Index). Rabbits were executed and liver tissues were taken for pathological staging of liver fibrosis (grouped by pathological stage into S0 group, S1 group, S2 group, S3 group and S4 group). In addition, the collagen I (Col I) and collagen III (Col III) expression levels in liver tissue were detected by Western blot. Results: Except for KURT, there were significant differences among the other eleven characteristic quantities (P < 0.05). LF Index, Col I and Col III expression levels showed a rising trend with increased pathological staging of liver fibrosis, presenting a positive correlation with the pathological staging of liver fibrosis (r = 0.718, r = 0.693, r = 0.611, P < 0.05). Conclusion: RTE quantitative analysis is expected for noninvasive evaluation of the pathological staging of liver fibrosis. PMID:24955175

  18. 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. PMID:19587415

  19. 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. PMID:26519771

  20. Detection and absolute quantitation of Tomato torrado virus (ToTV) by real time RT-PCR.

    PubMed

    Herrera-Vásquez, José Angel; Rubio, Luis; Alfaro-Fernández, Ana; Debreczeni, Diana Elvira; Font-San-Ambrosio, Isabel; Falk, Bryce W; Ferriol, Inmaculada

    2015-09-01

    Tomato torrado virus (ToTV) causes serious damage to the tomato industry and significant economic losses. A quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) method using primers and a specific TaqMan(®) MGB probe for ToTV was developed for sensitive detection and quantitation of different ToTV isolates. A standard curve using RNA transcripts enabled absolute quantitation, with a dynamic range from 10(4) to 10(10) ToTV RNA copies/ng of total RNA. The specificity of the RT-qPCR was tested with twenty-three ToTV isolates from tomato (Solanum lycopersicum L.), and black nightshade (Solanum nigrum L.) collected in Spain, Australia, Hungary and France, which covered the genetic variation range of this virus. This new RT-qPCR assay enables a reproducible, sensitive and specific detection and quantitation of ToTV, which can be a valuable tool in disease management programs and epidemiological studies. PMID:25956672

  1. Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR.

    PubMed

    Müller, C Catharina; Nourse, Jamie P; Nguyen, Tam H; Crane, Denis I

    2009-06-30

    The Cre/loxP-system has become an invaluable tool for the generation of tissue-specific gene disruption in mice. However, because Cre recombinase excision of individual genes can be variable, an accurate and sensitive method is necessary to determine the ultimate level of gene disruption. The analysis of gene disruption is particularly difficult for tissue that has been fixed for (immuno)histochemical analysis with paraformaldehyde. Here, we describe a simple, rapid and cost effective method for measurement of gene disruption using quantitative real-time PCR, through application to the analysis of PEX13 gene disruption in a brain-specific PEX13 mouse mutant. We show that this general protocol is suitable for both normal and paraformaldehyde-fixed tissue. PMID:19422853

  2. Real-time cellular analysis for quantitative detection of functional Clostridium difficile toxin in stool.

    PubMed

    Huang, Bin; Li, Haijing; Jin, Dazhi; Stratton, Charles W; Tang, Yi-Wei

    2014-04-01

    Rapid and accurate diagnosis and monitoring of Clostridium difficile infection (CDI) is critical for patient care and infection control. We will briefly review current laboratory techniques for the diagnosis of CDI and identify aspects needing improvement. We will also introduce a real-time cellular analysis (RTCA) assay developed for the diagnosis and monitoring of CDI using electronic impedance to assess the cell status. The RTCA assay uses impedance measurement to detect minute physiological changes in cells cultured on gold microelectrodes embedded in glass substrates in the bottom of microtiter wells. This assay has been adapted for quantitative detection of C. difficile functional toxin directly from stool specimens. Compared to conventional techniques and molecular assays, the RTCA assay provides a valuable tool for the diagnosis of CDI as well as for the assessment of clinical severity and for monitoring therapeutic efficacies. PMID:24649817

  3. 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

  4. 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. PMID:26395288

  5. 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

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

    PubMed

    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 ΔC t, 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

  7. Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    PubMed Central

    Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

    2013-01-01

    Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management. PMID:23811499

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons.

    PubMed

    Xu, Wentao; Wang, Chenguang; Zhu, Pengyu; Guo, Tianxiao; Xu, Yuancong; Huang, Kunlun; Luo, Yunbo

    2016-04-21

    Techniques of isothermal amplification have recently made great strides, and have generated significant interest in the field of point-of-care detection. Nicking endonuclease-mediated isothermal amplification (NEMA) is an example of simple isothermal technology. In this paper, a real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons (SMB-NEMA) of improved specificity and sensitivity is described. First, we optimized the prohibition of de novo synthesis by choosing Nt·BstNBI endonuclease. Second, the whole genome was successfully amplified with Nt·BstNBI (6 U), betaine (1 M) and trehalose (60 mM) for the first time. Third, we achieved 10 pg sensitivity for the first time after adding a small molecular beacon that spontaneously undergoes a conformational change when hybridizing to target, and the practical test validated the assay's application. The small molecular beacon has a similar melting temperature to the reaction temperature, but is approximately 10 bp shorter than the length of a traditional molecular beacon. A new threshold regulation was also established for isothermal conditions. Finally, we established a thermodynamic model for designing small molecular beacons. This multistate model is more correct than the traditional algorithm. This theoretical and practical basis will help us to monitor SMB-NEMA in a quantitative way. In summary, our SMB-NEMA method allows the simple, specific and sensitive assessment of isothermal DNA quantification. PMID:27027375

  10. Reversible Fluorescent Nanoswitch Based on Carbon Quantum Dots Nanoassembly for Real-Time Acid Phosphatase Activity Monitoring.

    PubMed

    Qian, Zhaosheng; Chai, Lujing; Zhou, Qian; Huang, Yuanyuan; Tang, Cong; Chen, Jianrong; Feng, Hui

    2015-07-21

    A reversible fluorescence nanoswitch by integrating carbon quantum dots nanoassembly and pyrophosphate ion is developed, and a reliable real-time fluorescent assay for acid phosphatase (ACP) activity is established on the basis of the fluorescence nanoswitch. Carbon quantum dots (CQDs) abundant in carboxyl groups on the surface, nickel(II) ion and pyrophosphate ion comprise the fluorescent nanoswitch, which operates in the following way: the nanoassembly consisting of CQDs and nickel ions can be triggered by pyrophosphate ion serving as an external stimulus. At the same time, the fluorescence nanoswitch switches between two fluorescence states (OFF and ON) accompanying shifts in their physical states aggregation and disaggregation. Based on the nanoswitch, the introduction of ACP leads to breakdown of pyrophosphate ions into phosphate ions and resultant fluorescence quenching due to catalytic hydrolysis of ACP toward pyrophosphate ions (PPi). Quantitative evaluation of ACP activity in a broad range from 18.2 U/L to 1300 U/L, with a detection limit of 5.5 U/L, can be achieved in this way, which endows the assay with sufficiently high sensitivity for practical detection in human serum and seminal plasma. PMID:26115095

  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. 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.

  13. 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.

  14. 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.

  15. 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. PMID:25798897

  16. 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

  17. 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

    2016-01-01

    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

  18. 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. PMID:26772407

  19. 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

  20. Reference gene selection for gene expression studies in lily using quantitative real-time PCR.

    PubMed

    Zhang, M F; Liu, Q; Jia, G X

    2016-01-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is an important technology used to analyze gene-expression levels. Reference genes, which are assumed to be expressed consistently across various developmental stages and in different tissues, were selected for expression level analysis. Using digital gene expression technology, we selected nine reference genes (18S, EF, CYCOL, SAND, GAPDH, ACTIN, BHLH, TIP, and Clathrin) as candidate reference genes for further study. Using three different analysis methods (GeNorm, NormFinder, and BestKeeper), a total of 144 lily (Lilium x formolongi "Raizan 3") samples were analyzed. The samples were collected from four different tissues under various developmental stages. In addition, leaves treated with different plant hormones were collected and analyzed. The data showed that the stability of the nine reference genes differed among samples, but TIP, EF, Clathrin, and BHLH could be identified as the most stable genes overall. In addition, the relative expression level of LfFT in different lily tissues with the competence to flower was also analyzed to verify the selected reference genes. This study constitutes an important source for selecting reference genes when analyzing the expression patterns of flowering time and floral development regulation genes in lily cultivars. PMID:27173307

  1. Development of Quantitative Real-time PCR Assays for Different Clades of "Candidatus Accumulibacter".

    PubMed

    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% (R(2) = 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

  2. Looking for reference genes for real-time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae).

    PubMed

    Majerowicz, D; Alves-Bezerra, M; Logullo, R; Fonseca-de-Souza, A L; Meyer-Fernandes, J R; Braz, G R C; Gondim, K C

    2011-12-01

    Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation. PMID:21929722

  3. A quantitative real-time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae.

    PubMed

    Bergmann, Nina; Fricke, Birgit; Schmidt, Martina C; Tams, Verena; Beining, Katrin; Schwitte, Hildegard; Boettcher, Anne A; Martin, Daniel L; Bockelmann, Anna-Christina; Reusch, Thorsten B H; Rauch, Gisep

    2011-11-01

    The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real-time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species-specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects. PMID:21777400

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

    PubMed

    Smith, Matthew M; Schmutz, Joel; Apelgren, Chloe; Ramey, Andrew M

    2015-04-01

    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 (R(2)=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. PMID:25655779

  5. 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.

  6. 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. PMID:25791491

  7. 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. PMID:25714139

  8. 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

  9. 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

  10. Analysis of gene expression in emerald ash borer (Agrilus planipennis) using quantitative real time-PCR.

    PubMed

    Bhandary, Binny; Rajarapu, Swapna Priya; Rivera-Vega, Loren; Mittapalli, Omprakash

    2010-01-01

    Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1(st)-, 2(nd)-, 3(rd)-, 4(th)-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium. PMID:20445495

  11. 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

  12. 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

  13. Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay.

    PubMed

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

    2012-03-01

    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 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) 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

  14. 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. PMID:24048535

  15. Robust Real-Time Reverse Transcription-PCR for Detection of Foot-and-Mouth Disease Virus Neutralizing Carryover Contamination

    PubMed Central

    Hwang, Ji-Hyeon; Shin, Yong-Keol; Park, So-Yeon; Kim, Jeesoo; Kim, Su-Mi; Kim, Byounghan; Park, Jong-Hyeon; Lee, Jong-Soo

    2015-01-01

    During an outbreak of foot-and-mouth disease (FMD), real-time reverse transcription-PCR (rRT-PCR) is the most commonly used diagnostic method to detect viral RNA. However, while this assay is often conducted during the outbreak period, there is an inevitable risk of carryover contamination. This study shows that the carryover contamination can be prevented by the use of target-specific restriction endonuclease in that assay. PMID:26560537

  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. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection. Conclusions FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA. PMID:23136846

  18. 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. PMID:25903566

  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. 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. PMID:24057254

  1. Rapid detection of Newcastle disease virus replication in embryonated chicken eggs using quantitative real time polymerase chain reaction.

    PubMed

    Gopinath, V P; Raj, Gopal Dhinakar; Raja, A; Kumanan, K; Elankumaran, Subbiah

    2011-01-01

    Newcastle disease virus (NDV), an avian paramyxovirus, is an economically important disease of poultry globally. Rapid methods to detect and differentiate the virus are important to curtail the spread of this virus. Nucleic acid based detection methods are routinely employed for diagnosis that suffer from the disadvantage of failure to discriminate viable virus and non-infectious genome. However, virus isolation remains the gold standard for diagnosis of field outbreaks. The sensitivity of virus isolation was combined with nucleic acid based detection methods so that the time taken for confirmatory diagnosis could be considerably reduced while increasing sensitivity. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and conventional RT-PCR techniques were compared for the detection of NDV genome replication in 9-11-day-old embryonated chicken eggs (ECE) using the nucleoprotein (NP) gene of the virus as a target. The results suggest that at least two to fourfold increase in cycle threshold (C(t)) values over the baseline C(t) value of samples lacking infectious virus, would indicate live NDV replication. The limit of detection of NDV replication using qRT-PCR was 1×10(4.0) mean embryo infective doses (EID(50)). The earliest time point when live virus replication was detectable by qRT-PCR or RT-PCR was 30h post-inoculation in ECE. PMID:20951166

  2. 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.

  3. 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. PMID:24518276

  4. 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...

  5. 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 ...

  6. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam (Mercenaria mercenaria)▿

    PubMed Central

    Liu, Qianqian; Allam, Bassem; Collier, Jackie L.

    2009-01-01

    We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples. PMID:19465523

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Multiplex real-time quantitative PCR methodology to assist in the breeding of potato lines with resistance to Verticillium wilt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato early dying (PED), caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide and PED-resistant cultivars currently represent only a small percentage of potato production. In this study we developed a real-time quantitative PCR (Q-PCR) approach to detect and quan...

  19. EVALUATION OF 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 (trademark)) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glab...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  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. 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.

  4. Real-time quantitative phase imaging based on transport of intensity equation with dual simultaneously recorded field of view.

    PubMed

    Tian, Xiaolin; Yu, Wei; Meng, Xin; Sun, Aihui; Xue, Liang; Liu, Cheng; Wang, Shouyu

    2016-04-01

    Since quantitative phase distribution reflects both cellular shapes and conditions from another view, compared to traditional intensity observation, different quantitative phase microscopic methods are proposed for cellular detections. However, the transport of intensity equation-based approach not only presents phase, but also intensity, which attracts much attention. While classical transport of intensity equation needs multi-focal images which often cannot realize simultaneous phase measurement, in this Letter, to break through the limitation, a real-time quantitative phase imaging method using transport of intensity equation is proposed. Two identical CCD cameras are set at the binocular tubes to capture the same field of view but at different focal planes. With a double-frame algorithm assuming that the on-focal image is the average of over- and under-focal information, the proposed method is capable of calculating quantitative phase distributions of samples accurately and simultaneously indicating its potentialities in cellular real-time monitoring. PMID:27192253

  5. 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. PMID:21803916

  6. 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

  7. Real-time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in myeloma.

    PubMed

    Compagno, Mara; Mantoan, Barbara; Astolfi, Monica; Boccadoro, Mario; Ladetto, Marco

    2005-01-01

    The evaluation of minimal residual disease (MRD) is critical in the evaluation of treatments aimed at maximal cytoreduction in multiple myeloma (MM). Qualitative evaluation of MRD now has a 10-yr-long history, but it remains a relatively sophisticated procedure. More recently, real-time quantitative approaches have also been developed. These approaches allow a very effective monitoring of disease but introduce additional complexity and costs to the procedure. This chapter describes how we currently perform real-time polymerase chain reaction (PCR) in MM. Compared to the first description of the assay in June 2000, significant improvements have been made. Although real-time PCR is the main focus of the chapter, most of the information suitable for a proper setup of a qualitative approach is also provided. PMID:15968100

  8. 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

  9. 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. PMID:27242878

  10. Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: the balance between accuracy and precision.

    PubMed

    Nordgård, Oddmund; Kvaløy, Jan Terje; Farmen, Ragne Kristin; Heikkilä, Reino

    2006-09-15

    Real-time reverse transcription polymerase chain reaction (RT-PCR) has gained wide popularity as a sensitive and reliable technique for mRNA quantification. The development of new mathematical models for such quantifications has generally paid little attention to the aspect of error propagation. In this study we evaluate, both theoretically and experimentally, several recent models for relative real-time RT-PCR quantification of mRNA with respect to random error accumulation. We present error propagation expressions for the most common quantification models and discuss the influence of the various components on the total random error. Normalization against a calibrator sample to improve comparability between different runs is shown to increase the overall random error in our system. On the other hand, normalization against multiple reference genes, introduced to improve accuracy, does not increase error propagation compared to normalization against a single reference gene. Finally, we present evidence that sample-specific amplification efficiencies determined from individual amplification curves primarily increase the random error of real-time RT-PCR quantifications and should be avoided. Our data emphasize that the gain of accuracy associated with new quantification models should be validated against the corresponding loss of precision. PMID:16899212

  11. 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. PMID:26327538

  12. 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.

  13. 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

  14. Development of Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction Assays to Detect Tembusu Virus in Culex tarsalis Mosquitoes

    PubMed Central

    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.

    2014-01-01

    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. PMID:25114013

  15. 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

  16. 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

    2016-01-01

    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

  17. Real-time cell analysis--a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.

    PubMed

    Teng, Zheng; Kuang, Xiaozhou; Wang, Jiayu; Zhang, Xi

    2013-11-01

    A newly developed electronic cell sensor array--the xCELLigence real-time cell analysis (RTCA) system is tested currently for dynamic monitoring of cell attachment, proliferation, damage, and death. In this study, human enterovirus (HEV71) infection of human rhabdomyosarcoma (RD) was used as an in vitro model to validate the application of this novel system as a straightforward and efficient assay for quantitative measurement of infectious viruses based on virus-induced cytopathic effect (CPE). Several experimental tests were performed including the determination of optimal seeding density of the RD cells in 96-well E-plates, RTCA real-time monitoring of the virus induced CPE and virus titer calculation, and viral neutralization test to determine HEV71 antibody titer. Traditional 50% tissue culture infective dose (TCID50) assay was also conducted for methodology comparison and validation, which indicated a consistent result between the two assays. These findings indicate that the xCELLigence RTCA system can be a valuable addition to current viral assays for quantitative measurement of infectious viruses and quantitation of neutralization antibody titer in real-time, warranting for future research and exploration of applications to many other animal and human viruses. PMID:23835032

  18. Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.

    PubMed

    Zhao, Wenjing; Li, Yan; Gao, Pengfei; Sun, Zhihong; Sun, Tiansong; Zhang, Heping

    2011-09-01

    Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang. PMID:21104423

  19. Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR▿ †

    PubMed Central

    Monnet, Christophe; Correia, Karine; Sarthou, Anne-Sophie; Irlinger, Françoise

    2006-01-01

    The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 105 to 1010 CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 105 CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses. PMID:16950905

  20. 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.

  1. 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. PMID:24447445

  2. Optimization of a duplex real-time PCR method for relative quantitation of infectious laryngotracheitis virus.

    PubMed

    Vagnozzi, Ariel; Riblet, Sylva M; Zavala, Guillermo; García, Maricarmen

    2012-06-01

    Infectious laryngotracheitis is a highly contagious respiratory disease of chickens controlled by biosecurity and vaccination with live attenuated or recombinant vaccines. Infectious laryngotracheitis virus (ILTV) infections are characterized by a peak of viral replication in the trachea followed by a steady decrease in replication that results in the establishment of latency. Estimation of viral load is an important tool to determine the stage of ILTV infection. Here, a multiplex real-time PCR was optimized for the quantification of ILTV genomes. Quantification of viral genomes was based on the amplification of the ILTV UL44 gene, and sample variability was normalized using the chicken (Gallusgallus domesticus) alpha2-collagen gene as an endogenous control in a duplex reaction. PMID:22856202

  3. Rapid and efficacious real-time quantitative PCR assay for quantitation of human DNA in forensic samples.

    PubMed

    Tringali, G; Barbaro, A; Insirello, E; Cormaci, P; Roccazzello, A M

    2004-12-01

    A reliable quantification of human DNA is necessary in the process of routine forensic human identification. When DNA is degraded, contaminated or associated with inhibitors, is important to accurately estimate the concentration of extracted DNA prior to perform an analysis based on nuclear markers amplified. In this work, a new approaches to DNA quantification employees the use of a real-time fluorescence probe system. The real-time PCR assay is highly sensitive, specific, rapid, cost-effective and flexible assay for analysis of forensic casework samples, can perform with DNA of poor quality and detect specifically amplifiable DNA rather than total DNA for STR analysis. PMID:15639571

  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. 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. PMID:22807508

  6. 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.

  7. 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., III; 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.

  8. 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.

  9. 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. PMID:25940928

  10. 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. PMID:20087729

  11. Quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons.

    PubMed

    Svilar, David; Vens, Conchita; Sobol, Robert W

    2012-01-01

    We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET). The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors. PMID:22895410

  12. A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV)

    PubMed Central

    2013-01-01

    Background Wheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases. Methods In this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and β-actin were selected in order to adjust the veracity of the real-time RT-PCR assay. Results We developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: β-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV. Conclusions The Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods. PMID:23725024

  13. 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. PMID:12083257

  14. 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. PMID:27468284

  15. 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

  16. 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

  17. 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

  18. Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

    PubMed

    Haase, Ilka; Brüning, Philipp; Matissek, Reinhard; Fischer, Markus

    2013-04-10

    Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated. PMID:23495652

  19. 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. PMID:14689155

  20. Quantitative real-time PCR (qPCR)--based tool for detection and quantification of Cordyceps militaris in soil.

    PubMed

    Saragih, Syaiful Amri; Takemoto, S; Hisamoto, Y; Fujii, M; Sato, H; Kamata, N

    2015-01-01

    A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems. PMID:25446034

  1. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes

    PubMed Central

    2013-01-01

    Background Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology–which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa. Results The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes. Conclusion We have developed a rapid, sensitive and specific rRT – PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection. PMID:24148652

  2. 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.

  3. 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

  4. Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T cell clones in mycobacterial infection

    PubMed Central

    Du, George; Qiu, Liyou; Shen, Ling; Sehgal, Probhat; Shen, Yun; Huang, Dan; Letvin, Norman L.; Chen, Zheng W.

    2010-01-01

    Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based real-time quantitation methods to quantitate numerous antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vβ families and 13 Jβ segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNγ-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10−5 to 10−6 antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by ≥2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNγ-producing T cell clones using as few as 2×106 PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases. PMID:16403511

  5. Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species.

    PubMed

    Xia, Qian-Feng; Liu, Jin-Bo; Liu, Ping; Qin, Xi; Qian, Shi-Yun; Tu, Zhi-Guang

    2012-01-01

    We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species. PMID:21964617

  6. 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. PMID:21873818

  7. 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

  8. Detection and quantitation of Citrus leaf blotch virus by TaqMan real-time RT-PCR.

    PubMed

    Ruiz-Ruiz, Susana; Ambrós, Silvia; Vives, María del Carmen; Navarro, Luis; Moreno, Pedro; Guerri, José

    2009-09-01

    A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times more sensitive than detection by conventional RT-PCR. An external standard curve using in vitro synthesized RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range (seven logarithmic units of concentration) and very low variation coefficient values. This protocol enabled detection of as little as 100 copies of CLBV RNA in various tissues and citrus varieties infected with CLBV sources from different geographical origins. The new assay greatly improves current detection methods for CLBV and it has been most helpful for the Spanish citrus sanitation, quarantine and certification programs, and fitness evaluation of infectious cDNA clones of CLBV, useful potentially as viral vectors for citrus. PMID:19406167

  9. Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

    PubMed Central

    Joly, Philippe; Falconnet, Pierre-Alain; André, Janine; Weill, Nicole; Reyrolle, Monique; Vandenesch, François; Maurin, Max; Etienne, Jerome; Jarraud, Sophie

    2006-01-01

    Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >103 CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory. PMID:16597985

  10. 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

  11. Real-time quantitation of Cu(II) by a fluorescence-based biosensing approach

    NASA Astrophysics Data System (ADS)

    Thompson, Richard B.; Zeng, Hui-Hui; Maliwal, Badri P.; Fierke, Carol A.

    2001-05-01

    Recently, there has been substantial interest in reducing the levels of toxic heavy metals in wastewater effluents from activities such as shipyards. Of particular interest is copper, which comprises tens of percent by weight of the hundreds of pounds of antifouling paint coating the bottom of a large vessel, but which is toxic to commercially important shellfish at sub-part per billion levels. As a result wastewater effluents must be monitored closely with sensor(s) capable of rapidly and accurately detecting excess copper in time to prevent release. We have pursued a fluorescence-based biosensing approach to obtain sub-ppb sensitivity for Cu(II) and immunity from interference from other cations abundant in sea water, such as Ca, Mg, and Sr. Our approach uses a protein, apocarbonic anhydrase II, as a very sensitive and selective ligand for Cu(II) which transduces the (reversible) binding of the metal as a change in fluorescence intensity, lifetime, or anisotropy, the first two of which may be conveniently measured through optical fiber. Thus we have been able to measure sub-ppb levels of Cu added to sea water, and to characterize the speciation of the Cu(II) to some degree, due to the presence of other ligands.

  12. 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

  13. Development of a quantitative real-time PCR for the detection of Tenacibaculum maritimum and its application to field samples.

    PubMed

    Fringuelli, E; Savage, P D; Gordon, A; Baxter, E J; Rodger, H D; Graham, D A

    2012-08-01

    The development and the application of a quantitative real-time PCR for the detection of Tenacibaculum maritimum are described. A set of primers and probe was designed to amplify a 155-bp fragment specific to the T. maritimum 16S rRNA gene. The test was shown to be very sensitive, able to detect as little as 4.8 DNA copies number μL(-1) . In addition, the assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R(2)  = 0.999) extending over 6 log(10) dilutions and a high efficiency (100%). The assay was applied to DNA samples extracted from 48 formalin-fixed paraffin-embedded (FFPE) Atlantic salmon, Salmo salar, gill tissues showing varying degrees of gill pathology (scored 0-3) and from 26 jellyfish samples belonging to the species Phialella quadrata and Muggiaea atlantica. For each sample, the bacterial load was normalised against the level of the salmonid elongation factor alpha 1 (ELF) detected by a second real-time PCR using previously published primers and probe. Tenacibaculum maritimum DNA was detected in 89% of the blocks with no signs of gill disease as well as in 95% of the blocks with mild-to-severe gill pathology. Association between bacterial load and gill pathology severity was investigated. T. maritimum DNA was detected at low level in four of the 26 jellyfish tested. PMID:22724390

  14. 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. PMID:19446978

  15. The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB) species.

    PubMed

    Penna, Antonella; Antonella, Penna; Galluzzi, Luca; Luca, Galluzzi

    2013-10-01

    In the last decade, various molecular methods (e.g., fluorescent hybridization assay, sandwich hybridization assay, automatized biosensor detection, real-time PCR assay) have been developed and implemented for accurate and specific identification and estimation of marine toxic microalgal species. This review focuses on the recent quantitative real-time PCR (qrt-PCR) technology developed for the control and monitoring of the most important taxonomic phytoplankton groups producing biotoxins with relevant negative impact on human health, the marine environment, and related economic activities. The high specificity and sensitivity of the qrt-PCR methods determined by the adequate choice of the genomic target gene, nucleic acid purification protocol, quantification through the standard curve, and type of chemical detection method make them highly efficient and therefore applicable to harmful algal bloom phenomena. Recent development of qrt-PCR-based assays using the target gene of toxins, such as saxitoxin compounds, has allowed more precise quantification of toxigenic species (i.e., Alexandrium catenella) abundance. These studies focus only on toxin-producing species in the marine environment. Therefore, qrt-PCR technology seems to offer the advantages of understanding the ecology of harmful algal bloom species and facilitating the management of their outbreaks. PMID:23247526

  16. 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.

  17. Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

    PubMed

    Chase, Dorothy M; Elliott, Diane G; Pascho, Ronald J

    2006-07-01

    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. PMID:16921877

  18. Quantitative real-time detection of carcinoembryonic antigen (CEA) from pancreatic cyst fluid using 3-D surface molecular imprinting.

    PubMed

    Yu, Yingjie; Zhang, Qi; Buscaglia, Jonathan; Chang, Chung-Chueh; Liu, Ying; Yang, Zhenhua; Guo, Yichen; Wang, Yantian; Levon, Kalle; Rafailovich, Miriam

    2016-07-21

    In this study, a sensitive, yet robust, biosensing system with real-time electrochemical readout was developed. The biosensor system was applied to the detection of carcinoembryonic antigen (CEA), which is a common marker for many cancers such as pancreatic, breast, and colon cancer. Real time detection of CEA during a medical procedure can be used to make critical decisions regarding further surgical intervention. CEA was templated on gold surface (RMS roughness ∼3-4 nm) coated with a hydrophilic self-assembled monolayer (SAM) on the working electrode of an open circuit potentiometric network. The subsequent removal of template CEA makes the biosensor capable of CEA detection based on its specific structure and conformation. The molecular imprinting (MI) biosensor was further calibrated using the potentiometric responses in solutions with known CEA concentrations and a detection limit of 0.5 ng ml(-1) was achieved. Potentiometric sensing was then applied to pancreatic cyst fluid samples obtained from 18 patients when the cyst fluid was also evaluated using ELISA in a certified pathology laboratory. Excellent agreement was obtained between the quantitation of CEA obtained by both the ELISA and MI biosensor detection for CEA. A 3-D MI model, using the natural rms roughness of PVD gold layers, is presented to explain the high degree of sensitivity and linearity observed in those experiments. PMID:27193921

  19. 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.

  20. 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.

  1. 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. PMID:26614294

  2. Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue

    PubMed Central

    Zhang, Wan-Xia; Fan, Jie; Ma, Jing; Rao, Yi-Song; Zhang, Li; Yan, You-E

    2016-01-01

    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. PMID:27338366

  3. Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

    PubMed Central

    Jothikumar, Narayanan; Cromeans, Theresa L.; Hill, Vincent R.; Lu, Xiaoyan; Sobsey, Mark D.; Erdman, Dean D.

    2005-01-01

    A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples. PMID:15933012

  4. Quantitative real-time PCR and phase specific serology are mutually supportive in Q fever diagnostics in goats.

    PubMed

    Sting, Reinhard; Molz, Kerstin; Philipp, Werner; Bothe, Friederike; Runge, Martin; Ganter, Martin

    2013-12-27

    This study presents results of quantitative pathogen detection by real-time PCR (qPCR) and phase-specific serology for complete Q fever diagnostics. For this, samples of 42 goats in total were taken during a Q fever outbreak. In the early phase of the Q-fever infection, 10(4)-10(8)Coxiella (C.) burnetii pathogens per vaginal swab and 10(2)-10(6)C. burnetii per ml milk were detected using quantitative real-time PCR (qPCR). Pathogen excretion decreased continuously within two months to less than 10(4) (vaginal swab) and 10(2) (milk) C. burnetii. At the end of the study there was a shift toward a 10 fold higher excretion of the pathogen via the genital tract and milk. At the start of the study, serological tests showed a dominance of the phase-2 antibody in 76% (22/29) of the goats in the MONA- (Multiple of Normal Activity) ELISA and 79% (23/29) in the IDEXX-ELISA, which was replaced by a phase-1 dominance in 85% (29/34) and 62% (21/34), of the animals respectively at the end of the study. Serum samples from 13 goats before lambing that excreted C. burnetii after lambing showed antibodies against phase 2 of 100% using MONA-ELISA and 77% in the IDEXX-ELISA. The most important diagnostic instrument for Q-fever infection in goats following birth is testing of vaginal swabs using qPCR. Phase-specific serology allows an estimation of possible pathogen excretion even before birth, as well as achieving valuable results for determination of the infection phase. PMID:24095624

  5. Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6

    PubMed Central

    Wakeley, P. R.; Johnson, N.; McElhinney, L. M.; Marston, D.; Sawyer, J.; Fooks, A. R.

    2005-01-01

    Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus. PMID:15956398

  6. 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. PMID:26025458

  7. Use of quantitative real-time PCR for direct detection of serratia marcescens in marine and other aquatic environments.

    PubMed

    Joyner, Jessica; Wanless, David; Sinigalliano, Christopher D; Lipp, Erin K

    2014-03-01

    Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health. PMID:24375136

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes.

    PubMed

    Chao, Day-Yu; Davis, Brent S; Chang, Gwong-Jen J

    2007-02-01

    A multiplex real-time reverse transcriptase PCR has been developed for the rapid detection and identification of eight medically important flaviviruses from laboratory-reared, virus-infected mosquito pools. The method used involves the gene-specific amplification of yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively) by use of the flavivirus consensus amplimers located at the RNA-dependent RNA polymerase domain of nonstructural protein 5. Virus-specific amplicons were detected by four newly characterized TaqMan fluorogenic probes (probes specific for YFV, JEV, WNV, and SLEV) and four previously published probes specific for DENV-1 to -4 (L. J. Chien, T. L. Liao, P. Y. Shu, J. H. Huang, D. J. Gubler, and G. J. Chang, J. Clin. Microbiol. 44:1295-1304, 2006). This assay had a specificity of 100% and various sensitivities of at least 3.5 PFU/ml for YFV, 2.0 PFU/ml for JEV, 10.0 PFU/ml for WNV, and 10.0 PFU/ml for SLEV. Additionally, we have developed an in vitro transcription system to generate RNase-resistant RNA templates for each of these eight viruses. These templates can be incorporated into the assay as RNA copy number controls and/or as external controls for RNA-spiked mosquito pools for quality assurance purposes. Although further study with mosquitoes collected in the field is needed, the incorporation of this assay into mosquito surveillance could be used as an early-warning system for the detection of medically important flaviviruses, particularly when the cocirculation of multiple viruses in the same region is suspected. PMID:17108075

  13. 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

  14. Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

    PubMed Central

    2010-01-01

    Background The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. Results We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII) transgene as well as of an internal control (β-actin), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus. Conclusions We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot plants. This method can be

  15. Quantitation of Taura syndrome virus by real-time RT-PCR with a TaqMan assay.

    PubMed

    Tang, Kathy F J; Wang, Jun; Lightner, Donald V

    2004-01-01

    A real-time RT-PCR assay was developed using a TaqMan probe to detect and quantify Taura syndrome virus (TSV) in penaeid shrimp. A pair of RT-PCR primers, which amplify a 72 bp DNA fragment, and a TaqMan probe were selected from open reading frame 1 (ORF1) of the TSV genome. The primers and TaqMan probe used in this assay reacted with TSV isolates from Hawaii, Texas, Colombia, Mexico, Belize, Indonesia, and Thailand, but neither with RNA of healthy shrimp nor with an isolate of yellow head virus. A plasmid (pTSV-1) that contains the target TSV sequence was constructed and used to generate positive control RNA through in vitro transcription. The positive control RNA was used to demonstrate that the real-time RT-PCR assay has a detection limit of 100 copies and a log-linear range up to 10(8) copies of TSV RNA. This quantitative method was found to be highly reproducible, with low intra- and inter-assay variation. Coefficient of variation (CVs) values were 0.04-8.9 and 0.05-3.7%, respectively, for replicates within and among assays. This assay was used to quantify TSV in both acutely and chronically infected shrimp in a laboratory experiment. The quantities of TSV in the tissues of pleopods and gills were not significantly different, and there was no difference in TSV levels between the acutely and chronically infected groups. However, in the chronically infected shrimp, the quantities of TSV were one to two orders of magnitude higher in the lymphoid organ than in either gills or pleopods. This assay proved to be specific with high sensitivity, and it can be used to detect and quantify TSV in shrimp samples. PMID:14656468

  16. 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

  17. 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

  18. 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. PMID:26141544

  19. Clinical value of real-time elastography quantitative parameters in evaluating the stage of liver fibrosis and cirrhosis

    PubMed Central

    GE, LAN; SHI, BAOMIN; SONG, YE; LI, YUAN; WANG, SHUO; WANG, XIUYAN

    2015-01-01

    The aim of the present study was to assess the value of real-time elastography (RTE) quantitative parameters, namely the liver fibrosis (LF) index and the ratio of blue area (%AREA), in evaluating the stage of liver fibrosis. RTE quantitative analysis software was used to examine 120 patients with chronic hepatitis in order to obtain the values for 12 quantitative parameters from the elastic images. The diagnostic performance of two such parameters, the LF index and %AREA, were assessed with a receiver operating characteristic (ROC) curve to determine the optimal diagnostic cut-off values for liver cirrhosis and fibrosis. A good correlation was observed between the LF index and %AREA with the fibrosis stage. The areas under the ROC curve for the LF index were 0.985 for the diagnosis of liver cirrhosis and 0.790 for liver fibrosis. With regard to %AREA, the areas under the ROC curve for the diagnosis of liver cirrhosis and fibrosis were 0.963 and 0.770, respectively. An LF index of >3.25 and a %AREA of >28.83 for the diagnosis of cirrhosis stage resulted in sensitivity values of 100 and 100%, specificity values of 88.9 and 85.9% and accuracy values of 90.8 and 88.3%, respectively. The LF index and %AREA parameters exhibited higher reliability in the diagnosis of liver cirrhosis compared with the diagnosis of the liver fibrosis stage. However, the two parameters possessed a similar efficacy in the diagnosis of liver cirrhosis and the stage of liver fibrosis. Therefore, the quantitative RTE parameters of the LF index and %AREA may be clinically applicable as reliable indices for the early diagnosis of liver cirrhosis, without the requirement of an invasive procedure. PMID:26622426

  20. Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza Virus by Real-Time Reverse Transcription Polymerase Chain Reaction Assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Comparison of pooling 11 or 5 oropharyngeal swabbings for detecting avian influenza virus by real-time reverse transcription-PCR in broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of pooling five or 11 orophyarngeal (O/P) swabbings on detecting avian influenza virus (AIV) by real-time reverse transcription-polymerase chain reaction (RRT-PCR) was evaluated. The model used for the evaluation was designed to minimize viral load and thus assess the effect of the pooli...

  2. Specific detection of rinderpest virus by real-time reverse transcription-PCR in preclincal and clinical samples of experimentally infected cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. 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...

  4. A Capsid Gene-Based Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Detection of Marine Vesiviruses in the Caliciviridae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens.

    PubMed

    Lee, Dae-Young; Lauder, Heather; Cruwys, Heather; Falletta, Patricia; Beaudette, Lee A

    2008-07-15

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10(3)A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater. PMID:18423816

  6. 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

  7. Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus.

    PubMed

    Ciulli, Sara; Pinheiro, Ana Cristina de Aguiar Saldana; Volpe, Enrico; Moscato, Michele; Jung, Tae Sung; Galeotti, Marco; Stellino, Sabrina; Farneti, Riccardo; Prosperi, Santino

    2015-03-01

    Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains. PMID:25522921

  8. Detection and identification of Rift Valley fever virus in mosquito vectors by quantitative real-time PCR.

    PubMed

    Mwaengo, D; Lorenzo, G; Iglesias, J; Warigia, M; Sang, R; Bishop, R P; Brun, A

    2012-10-01

    Diagnostic methods allowing for rapid identification of pathogens are crucial for controlling and preventing dissemination after disease outbreaks as well as for use in surveillance programs. For arboviruses, detection of the presence of virus in their arthropod hosts is important for monitoring of viral activity and quantitative information is useful for modeling of transmission dynamics. In this study, molecular detection of Rift Valley fever virus (RVFV) in mosquito samples from the 2006 to 2007 East African outbreaks was performed using quantitative real-time PCR assay (qRT-PCR). Specific RVFV sequence-based primer/fluorogenic (TaqMan) probe sets were derived from the L and S RNA segments of the virus. Both primer-probe L and S segment-based combinations detected genomic RVFV sequences, with generally comparable levels of sensitivity. Viral loads from three mosquito species, Aedes mcintoshi, Aedes ochraceus and Mansonia uniformis were estimated and significant differences of between 5- and 1000-fold were detected between Ae. mcintoshi and M. uniformis using both the L and S primer-probe-based assays. The genetic relationships of the viral sequences in mosquito samples were established by partial M segment sequencing and assigned to the two previously described viral lineages defined by analysis of livestock isolates obtained during the 2006-2007 outbreak, confirming that similar viruses were present in both the vector and mammalian host. The data confirms the utility of qRT-PCR for identification and initial quantification of virus in mosquito samples during RVFV outbreaks. PMID:22841800

  9. Uses of statistical editing of real-time ambulatory electrocardiographic recordings for quantitative ventricular ectopic beat counts.

    PubMed

    Crow, R S; Hannan, P; Jacobs, D R; Mascioli, S

    1992-04-01

    Real-time ambulatory monitoring analyzes each heart beat, counts events, and stores ECG samples for later visual verification. Typically, a physician examines these to determine whether the computer algorithm accurately identified arrhythmias. Physician editing is performed using best clinical judgement. We developed a simple statistical editing procedure for adjusting false positive and false negative computer detections. In 20 subjects having 24-hr monitoring we compared statistically edited ventricular premature beat (VPB) counts and pair/run counts with the unedited monitor counts and with physician assessment using a visual counted gold standard. The agreement of the statistically edited count with the visual standard was 65% for total VPB, 85% for VPB pair/runs, and 90% for a risk score based on ventricular ectopy. Corresponding agreements for unedited monitor count were 15, 25, and 30%, respectively. Physician assessment was not sufficiently precise to allow quantitative count estimates. This study indicates a statistical editing procedure substantially increases the level of agreement between the visual standard and the monitor count of VPB frequency and complexity. Statistically edited data are suitable for quantitative counts of VPB and other arrhythmic events in research and in medical diagnosis and treatment. This editing procedure can be a useful adjunct to any ambulatory monitoring system. PMID:1582190

  10. Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR.

    PubMed

    Collantes-Fernández, Esther; Zaballos, Angel; Alvarez-García, Gema; Ortega-Mora, Luis M

    2002-04-01

    We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10(-1) tachyzoite per assay was observed with excellent linearity (R(2) = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques (kappa = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection. PMID:11923330

  11. 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

  12. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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

    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. PMID:24648561

  14. 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

  15. A duplex quantitative real-time PCR assay for the detection of Haplosporidium and Perkinsus species in shellfish.

    PubMed

    Xie, Zhixun; Xie, Liji; Fan, Qing; Pang, Yaoshan; Deng, Xianwen; Xie, Zhi Qin; Liu, Jiabo; Khan, Mazhar I

    2013-04-01

    A duplex quantitative real-time polymerase chain reaction (dq-PCR) assay was optimized to simultaneously detect Haplosporidium spp. and Perkinsus spp. of shellfish in one reaction. Two sets of specific oligonucleotide primers for Haplosporidium spp. and Perkinsus spp., along with two hydrolysis probes specific for each parasite group, were used in the assay. The dq-PCR results were detected and analyzed using the Light Cycler 2.0 software system. The dq-PCR identified and differentiated the two protozoan parasite groups. The sensitivity of the dq-PCR assay was 200 template copies for both Haplosporidium spp. and Perkinsus spp. No DNA product was amplified when known DNA from Marteilia refringens, Toxoplasma gondii, Bonamia ostreae, Escherichia coli, Cymndinium spp., Mykrocytos mackini, Vibrio parahaemolyticus, and shellfish tissue were used as templates. A total of 840 oyster samples from commercial cultivated shellfish farms from two coastal areas in China were randomly collected and tested by dq-PCR. The detection rate of Haplosporidium spp. was 8.6% in the Qindao, Shandong coastal area, whereas Perkinsus spp. was 8.3% coastal oysters cultivated from shellfish farms of Beihai, Guangxi. The dqPCR results suggested that Haplosporidium spp. was prevalent in oysters from Qindao, Shandong, while Perkinsus spp. was prevalent in oysters from the coastal areas of Beihai, Guangxi. This dq-PCR could be used as a diagnostic tool to detect Haplosporidium spp. and Perkinsus spp. in cultivated shellfish. PMID:23371501

  16. Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment.

    PubMed

    Shannon, K E; Lee, D-Y; Trevors, J T; Beaudette, L A

    2007-08-15

    Bacteria were detected at five stages of municipal wastewater treatment using TaqMan(R) real-time quantitative PCR (qPCR). Thirteen probe and primer sets were tested for diverse pathogens that may be present in wastewater, including Aeromonas hydrophila, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, E. coli O157:H7, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella sp., and Staphylococcus aureus. The sensitivity of the assay was 100 fg of genomic DNA (=22 gene copies), based on a standard curve generated using A. hydrophila purified DNA. Samples from five stages of wastewater treatment were collected, including raw wastewater, primary effluents, mixed liquor, waste activated sludge and final effluents. In duplicate samples, E. coli, K. pneumoniae, C. perfringens and E. faecalis were detected throughout the wastewater process, and their numbers decreased by 3.52-3.98, 4.23-4.33, 3.15-3.39, and 3.24 orders of magnitude respectively, between the raw wastewater and final effluent stage. This qPCR method was effective for the detection of pathogens in wastewater and confirmed that the risk of exposure to pathogens in the wastewater discharge was well within the Environment Canada guidelines. PMID:17462712

  17. 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. PMID:24610857

  18. 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. PMID:27033773

  19. Reference Genes Selection for Quantitative Real-Time PCR Using RankAggreg Method in Different Tissues of Capra hircus

    PubMed Central

    Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Bakhtiarizadeh, Mohammad Reza

    2013-01-01

    Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, Capra hircus, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes. PMID:24358246

  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. PMID:22843326

  1. 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). PMID:27122388

  2. Evaluation of Virulence Gene Expression Patterns in Acinetobacter baumannii Using Quantitative Real-Time Polymerase Chain Reaction Array.

    PubMed

    Lannan, Ford M; O'conor, Daniel K; Broderick, Joseph C; Tate, Jamison F; Scoggin, Jacob T; Moran, Nicholas A; Husson, Christopher M; Hegeman, Erik M; Ogrydziak, Cole E; Singh, Sneha A; Vafides, Andrew G; Brinkley, Carl C; Goodin, Jeremy L

    2016-09-01

    According to the Centers for Disease Control's recently devised National Strategy for Combating Antibiotic-Resistant Bacteria, Acinetobacter baumannii is a "serious" threat level pathogen. A. baumannii's notoriety stems from the fact that a large number of modern strains are multidrug resistant and persist in the hospital setting, thus causing numerous deaths per year. It is imperative that research focus on a more fundamental understanding of the factors responsible for the success of A. baumannii. Toward this end, our group investigated virulence gene expression patterns in a recently characterized wound isolate, AB5075, using quantitative real-time polymerase chain reaction array. Notably, several genes showed statistically significant upregulation at 37°C compared to 25°C; MviM, Wbbj, CarO, and certain genes of the Bas, Bar, and Csu operons. Additionally, we found that in vitro biofilm formation by Csu transposon insertion mutant strains is attenuated. These findings validate previous reports that suggest a link between the Csu operon and biofilm formation. More importantly, our results demonstrate a successful method for evaluating the significance of previously identified virulence factors in a modern and clinically relevant strain of A. baumannii, thereby providing a path toward a more fundamental understanding of the pathogenicity of A. baumannii. PMID:27612361

  3. 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. PMID:26040611

  4. 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. PMID:21845960

  5. 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.

  6. 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. PMID:15859082

  7. 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

  8. Evaluation of putative reference genes for quantitative real-time PCR normalization in Lilium regale during development and under stress

    PubMed Central

    Zhang, Ming-Fang

    2016-01-01

    Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression, Lilium, and particularly L. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely 18S rRNA, ACT, BHLH, CLA, CYP, EF1, GAPDH, SAND and TIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms, BHLH was superior to the other candidates when all the experimental treatments were analyzed together; CLA and EF1 were also recommended by two of the three algorithms. As for specific conditions, EF1 under various developmental stages, SAND under biotic stress, CYP/GAPDH under drought stress, and TIP41 under salinity stress were generally considered suitable. All the algorithms agreed on the stability of SAND and GAPDH under cold stress, while only CYP was selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level of LrLOX in leaves inoculated with B. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding of Lilium. PMID:27019788

  9. Leptin in Whales: Validation and Measurement of mRNA Expression by Absolute Quantitative Real-Time PCR

    PubMed Central

    Ball, Hope C.; Holmes, Robert K.; Londraville, Richard L.; Thewissen, Johannes G. M.; Duff, Robert Joel

    2013-01-01

    Leptin is the primary hormone in mammals that regulates adipose stores. Arctic adapted cetaceans maintain enormous adipose depots, suggesting possible modifications of leptin or receptor function. Determining expression of these genes is the first step to understanding the extreme physiology of these animals, and the uniqueness of these animals presents special challenges in estimating and comparing expression levels of mRNA transcripts. Here, we compare expression of two model genes, leptin and leptin-receptor gene-related product (OB-RGRP), using two quantitative real-time PCR (qPCR) methods: “relative” and “absolute”. To assess the expression of leptin and OB-RGRP in cetacean tissues, we first examined how relative expression of those genes might differ when normalized to four common endogenous control genes. We performed relative expression qPCR assays measuring the amplification of these two model target genes relative to amplification of 18S ribosomal RNA (18S), ubiquitously expressed transcript (Uxt), ribosomal protein 9 (Rs9) and ribosomal protein 15 (Rs15) endogenous controls. Results demonstrated significant differences in the expression of both genes when different control genes were employed; emphasizing a limitation of relative qPCR assays, especially in studies where differences in physiology and/or a lack of knowledge regarding levels and patterns of expression of common control genes may possibly affect data interpretation. To validate the absolute quantitative qPCR methods, we evaluated the effects of plasmid structure, the purity of the plasmid standard preparation and the influence of type of qPCR “background” material on qPCR amplification efficiencies and copy number determination of both model genes, in multiple tissues from one male bowhead whale. Results indicate that linear plasmids are more reliable than circular plasmid standards, no significant differences in copy number estimation based upon background material used, and

  10. Leptin in whales: validation and measurement of mRNA expression by absolute quantitative real-time PCR.

    PubMed

    Ball, Hope C; Holmes, Robert K; Londraville, Richard L; Thewissen, Johannes G M; Duff, Robert Joel

    2013-01-01

    Leptin is the primary hormone in mammals that regulates adipose stores. Arctic adapted cetaceans maintain enormous adipose depots, suggesting possible modifications of leptin or receptor function. Determining expression of these genes is the first step to understanding the extreme physiology of these animals, and the uniqueness of these animals presents special challenges in estimating and comparing expression levels of mRNA transcripts. Here, we compare expression of two model genes, leptin and leptin-receptor gene-related product (OB-RGRP), using two quantitative real-time PCR (qPCR) methods: "relative" and "absolute". To assess the expression of leptin and OB-RGRP in cetacean tissues, we first examined how relative expression of those genes might differ when normalized to four common endogenous control genes. We performed relative expression qPCR assays measuring the amplification of these two model target genes relative to amplification of 18S ribosomal RNA (18S), ubiquitously expressed transcript (Uxt), ribosomal protein 9 (Rs9) and ribosomal protein 15 (Rs15) endogenous controls. Results demonstrated significant differences in the expression of both genes when different control genes were employed; emphasizing a limitation of relative qPCR assays, especially in studies where differences in physiology and/or a lack of knowledge regarding levels and patterns of expression of common control genes may possibly affect data interpretation. To validate the absolute quantitative qPCR methods, we evaluated the effects of plasmid structure, the purity of the plasmid standard preparation and the influence of type of qPCR "background" material on qPCR amplification efficiencies and copy number determination of both model genes, in multiple tissues from one male bowhead whale. Results indicate that linear plasmids are more reliable than circular plasmid standards, no significant differences in copy number estimation based upon background material used, and that the use of