New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

ABSTRACTLaboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

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Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.9.5624-5626.2005 ◽

2005 ◽

Vol 71 (9) ◽

pp. 5624-5626 ◽

Cited By ~ 34

Author(s):

X. C. Shan ◽

P. Wolffs ◽

M. W. Griffiths

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Quantitative Detection ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

Immunomagnetic Capture ◽

Capture Method

ABSTRACT In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02660-05 ◽

2006 ◽

Vol 72 (6) ◽

pp. 3846-3855 ◽

Cited By ~ 313

Author(s):

M. Isabel Costafreda ◽

Albert Bosch ◽

Rosa M. Pintï¿½

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.

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

Poultry Science ◽

10.3382/ps.2014-04024 ◽

2014 ◽

Vol 93 (9) ◽

pp. 2184-2192 ◽

Cited By ~ 18

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

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

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.4371-4374.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 4371-4374 ◽

Cited By ~ 34

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.

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