scholarly journals Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA

1999 ◽  
Vol 65 (1) ◽  
pp. 322-326 ◽  
Author(s):  
Charlotte Arnal ◽  
Virginie Ferre-Aubineau ◽  
Berangere Mignotte ◽  
Berthe Marie Imbert-Marcille ◽  
Sylviane Billaudel
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Internal Standard ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Reproducible Method ◽  
Dna Enzyme Immunoassay

ABSTRACT To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 104 to 107 copies of HAV/gram or 400 to 106 50% tissue culture infective doses/ml.

scholarly journals Development of a Reverse Transcription-PCR–DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish

2001 ◽  
Vol 67 (2) ◽  
pp. 742-749 ◽  
Author(s):  
Kellogg J. Schwab ◽  
Frederick H. Neill ◽  
Françoise Le Guyader ◽  
Mary K. Estes ◽  
Robert L. Atmar
Keyword(s):  
Enzyme Immunoassay ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Product ◽  
Dna Enzyme Immunoassay

ABSTRACT Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly “Norwalk-like” viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR–oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.

scholarly journals Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

2001 ◽  
Vol 67 (12) ◽  
pp. 5593-5600 ◽  
Author(s):  
Julie Jean ◽  
Burton Blais ◽  
André Darveau ◽  
Ismaı̈l Fliss
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Dot Blot ◽  
Reverse Transcription Pcr ◽  
Dot Blot Assay

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

scholarly journals Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples

2006 ◽  
Vol 72 (6) ◽  
pp. 3846-3855 ◽  
Author(s):  
M. Isabel Costafreda ◽  
Albert Bosch ◽  
Rosa M. Pint�
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Accurate Estimation ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Development Evaluation

ABSTRACT A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

Green Onions: Potential Mechanism for Hepatitis A Contamination

2006 ◽  
Vol 69 (6) ◽  
pp. 1468-1472 ◽  
Author(s):  
DAVID D. CHANCELLOR ◽  
SHACHI TYAGI ◽  
MICHAEL C. BAZACO ◽  
SARA BACVINSKAS ◽  
MICHAEL B. CHANCELLOR ◽  
...  
Keyword(s):  
Liquid Medium ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Potential Mechanism ◽  
Rt Pcr ◽  
Fluorescent Microspheres ◽  
Reverse Transcription Pcr ◽  
Hydroponically Grown

The largest documented foodborne hepatitis A outbreak in U.S. history occurred in November 2003. The source of that outbreak was green onions from a farm in Mexico. Two biomarkers were used to determine ways in which hepatitis A virus (HAV) can contaminate onions. Fluorescent microspheres (1.0 to 10 μm) and HAV vaccine were placed on the soil and the surfaces of pot-grown onions and in the liquid medium of hydroponically cultivated onions. Reverse transcription PCR (RTPCR) was used to identify HAV RNA. Microspheres were found on the outside and inside of the pot-grown onions for up to 60 days. RT-PCR revealed HAV RNA from the vaccine in well-washed green onions. In the hydroponically grown onions, microspheres were found throughout the onion after only 1 day. RT-PCR also revealed HAV RNA inside the hydroponically grown onions. Both biomarkers support the hypothesis that HAV can contaminate the inside of the growing onion and can be taken up intracellularly through the roots. Once inside, the particles are impossible to remove by cleaning.

scholarly journals Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

1999 ◽  
Vol 45 (9) ◽  
pp. 1397-1407 ◽  
Author(s):  
Alice Ylikoski ◽  
Minna Sjöroos ◽  
Åke Lundwall ◽  
Matti Karp ◽  
Timo Lövgren ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Internal Standard ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

Abstract Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males]. Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

scholarly journals A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater

2003 ◽  
Vol 69 (6) ◽  
pp. 3158-3164 ◽  
Author(s):  
G. Shay Fout ◽  
Beth C. Martinson ◽  
Michael W. N. Moyer ◽  
Daniel R. Dahling
Keyword(s):  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Disease Outbreaks ◽  
Enteric Viruses ◽  
The United States ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Groundwater Samples ◽  
Human Enteric Viruses

ABSTRACT Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.

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

2002 ◽  
Vol 68 (3) ◽  
pp. 1351-1357 ◽  
Author(s):  
Camile Pizeta Semighini ◽  
Mozart Marins ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Quantitative Analysis ◽  
Real Time ◽  
Aspergillus Nidulans ◽  
Abc Transporter ◽  
Reverse Transcription ◽  
Wild Type ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Reverse Transcription Pcr ◽  
Nitroquinoline Oxide

ABSTRACT 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.

Evaluation of a Sensitive Reverse Transcription PCR–Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis A Virus in Oysters (Saccostrea glomerata) on the East Coast of the Gulf of Thailand

2014 ◽  
Vol 77 (5) ◽  
pp. 859-863 ◽  
Author(s):  
URAIWAN INTAMASO ◽  
SITTHISAK KETKHUNTHOD
Keyword(s):  
Immunosorbent Assay ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
East Coast ◽  
Hepatitis A Virus ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gulf Of Thailand ◽  
Rt Pcr ◽  
The Gulf Of Thailand

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine–arginine–polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

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