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 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 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 Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4371-4374 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

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.

Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus

1998 ◽  
Vol 44 (3) ◽  
pp. 298-302 ◽  
Author(s):  
Charlotte Arnal ◽  
Virginie Ferré-Aubineau ◽  
Bernard Besse ◽  
Sylviane Billaudel
Keyword(s):  
Polymerase Chain Reaction ◽  
Enzyme Immunoassay ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Single Step ◽  
Chain Reaction ◽  
Complete Automation ◽  
Polymerase Chain ◽  
Dna Enzyme Immunoassay

Reverse transcription polymerase chain reaction, using either nested or seminested primers, is used extensively for the detection of viruses in small quantities. However, existing methods are prone to false positive reactions. We report here an improved polymerase chain reaction technique based on the use of longer primers (39 nucleotides) with single-step amplification, applied to the detection of hepatitis A in low quantities. While the sensitivity of this technique (10 x the 50% tissue culture infective dose) is equivalent to that of existing methods, it is a simpler procedure, less time consuming, and less susceptible to contamination and therefore provides a more reliable tool for routine diagnosis. Finally, the development of a DNA enzyme immunoassay detection technique and the complete automation of the procedure allow a large number of samples to be processed in clinical laboratories.Key words: long primers, hepatitis A virus, reverse transcription polymerase chain reaction, seminested PCR, DNA enzyme immunoassay.

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 Evaluation of a Commercial DNA Enzyme Immunoassay for Detection of Enterovirus Reverse Transcription-PCR Products Amplified from Cerebrospinal Fluid Specimens

2000 ◽  
Vol 38 (11) ◽  
pp. 4260-4261 ◽  
Author(s):  
Pampee P. Young ◽  
Richard S. Buller ◽  
Gregory A. Storch
Keyword(s):  
Cerebrospinal Fluid ◽  
Enzyme Immunoassay ◽  
Optical Density ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Clinical Laboratories ◽  
Pcr Products ◽  
Dna Enzyme Immunoassay ◽  
Culture Positive

We evaluated the DiaSorin DNA enzyme immunoassay (DEIA) kit for detection of enteroviral reverse transcription-PCR (RT-PCR) products amplified from cerebrospinal fluid. By use of an optical density of 0.05 as the absorbance cutoff, 35% of 198 specimens were PCR positive, whereas 16% were culture positive. DEIA was rapid and sensitive and can help implement enterovirus RT-PCR in clinical laboratories.

scholarly journals Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay

2014 ◽  
Vol 93 (9) ◽  
pp. 2184-2192 ◽  
Author(s):  
X.J. Wen ◽  
A.C. Cheng ◽  
M.S. Wang ◽  
R.Y. Jia ◽  
D.K. Zhu ◽  
...  
Keyword(s):  
Virus Type ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Duck Hepatitis ◽  
Type 3

Improvements in the RT-PCR detection of enteric viruses in environmental samples

1998 ◽  
Vol 38 (12) ◽  
pp. 83-86 ◽  
Author(s):  
K. J. Schwab ◽  
F. H. Neill ◽  
M. K. Estes ◽  
R. L. Atmar
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Environmental Samples ◽  
Internal Standard ◽  
Pcr Amplification ◽  
Enteric Viruses ◽  
New Method ◽  
Rt Pcr ◽  
Pcr Product ◽  
Enzyme Method

Current methods for the detection of nucleic acid from enteric viruses in environmental samples usually involve extensive concentration and purification of target viruses followed by RT-PCR amplification using two enzymes, reverse transcriptase and Taq polymerase. We have developed a modified method that improves RT-PCR assays by: (i) the use of an RT-PCR internal standard control RNA to identify potential false negative results caused by inhibition of RT-PCR enzymes; (ii) the use of rTth (Perkin-Elmer, Foster City, CA), a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase in a single-tube, single-buffer, elevated-temperature reaction; and (iii) the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentre Technologies, Madison, WI) to prevent PCR product carryover contamination. The new method was compared to the traditional two-enzyme, RT-PCR method for detection of Norwalk virus (NV) and hepatitis A virus (HAV) in buffer, stool, clam and oyster samples. The new method was at least as sensitive in NV and HAV detection compared to the traditional two-enzyme method. The internal standard control successfully detected inhibitors to RT-PCR amplification. NV and HAV PCR products generated with dUTP replacing dTTP during amplification were seeded into subsequent samples to test the prevention of PCR product carryover contamination by HK-UNG. The new method successfully eliminated PCR product carryover contamination in contrast to the traditional two-enzyme method. These improvements to viral nucleic acid detection have the potential to improve sensitivity, specificity and confidence in RT-PCR results.

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