scholarly journals Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 159
Author(s):  
Robert A. Kozak ◽  
Candace Rutherford ◽  
Melissa Richard-Greenblatt ◽  
N. Y. Elizabeth Chau ◽  
Ana Cabrera ◽  
...  
Keyword(s):  
Clinical Utility ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Health Concern ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Treatment And Prevention ◽  
Virus Assay ◽  
Stool Samples ◽  
Stool Specimens

Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10–30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5′-untranslated region (5′-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.

Survival and Persistence of Norovirus, Hepatitis A Virus, and Feline Calicivirus in Marinated Mussels

2004 ◽  
Vol 67 (8) ◽  
pp. 1743-1750 ◽  
Author(s):  
JOANNE HEWITT ◽  
GAIL E. GREENING
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Feline Calicivirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Potential Health ◽  
Infectious Dose ◽  
Log Reduction ◽  
Public Health Authorities

Noroviruses (NV) and hepatitis A virus (HAV) are foodborne enteric viruses associated with outbreaks of disease following consumption of raw or lightly cooked bivalve shellfish. Marinated mussels are a popular delicacy, but there is no published information on whether enteric viruses survive the marination process. The survival and persistence of HAV, NV, and a surrogate calicivirus, feline calicivirus (FCV), in marinated mussels over time was determined. NV, HAV, and FCV were inoculated into marinated mussels and marinade liquid and then held at 4°C for up to 4 weeks. Survival of HAV and FCV was quantified by determining the 50% tissue culture infectious dose (TCID50), and these results were correlated with those of the reverse transcription (RT)–PCR assay. The persistence of nonculturable NV was determined by RT–PCR assay only. Over 4 weeks, HAV survived exposure to acid marinade at pH 3.75. There was a 1.7-log reduction in HAV TCID50 titer but no reduction in NV or HAV RT-PCR titer after 4 weeks in marinated mussels. FCV was inactivated in acid conditions although it was still detectable by RT-PCR. To simulate preharvest virus contamination and commercial marination processing, experiments using fresh mussels infected with HAV and NV were performed. HAV and NV persistence was determined using semiquantitative real-time RT-PCR, and HAV infectivity was determined by the TCID50 assay. HAV retained infectivity following simulated commercial marination and exposure to acid conditions over 4 weeks. The survival of pathogenic enteric viruses in marinated mussels constitutes a potential health risk and so is of concern to public health authorities.

Improved duplex RT-PCR assay for differential diagnosis of mixed infection of duck hepatitis A virus type 1 and type 3 in ducklings

2013 ◽  
Vol 192 (1-2) ◽  
pp. 12-17 ◽  
Author(s):  
Lin-Lin Chen ◽  
Qian Xu ◽  
Rui-Hua Zhang ◽  
Lei Yang ◽  
Jing-Xin Li ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Virus Type ◽  
Mixed Infection ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Duck Hepatitis ◽  
Type 3

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.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

Polymerase chain reaction amplification and typing of rotavirus in environmental samples

1995 ◽  
Vol 31 (5-6) ◽  
pp. 371-374 ◽  
Author(s):  
R. Gajardo ◽  
R. M. Pintó ◽  
A. Bosch
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Polymerase Chain Reaction Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Group A ◽  
Reaction Amplification ◽  
Stool Specimens ◽  
Polymerase Chain

A reverse transcription polymerase chain reaction (RT-PCR) assay is described that has been developed for the detection and serotyping of group A rotavirus in stool specimens and concentrated and non-concentrated sewage specimens.

DETECTION AND DIFFERENTIATION OF THE VACCINE STRAIN AND FIELD ISOLATES OF DUCK HEPATITIS A VIRUS TYPE 1 USING REAL-TIME RT-PCR AND HIGH RESOLUTION MELTING ASSAYS

2015 ◽  
Vol 41 (04) ◽  
pp. 229-235
Author(s):  
Kuang-Po Li ◽  
Shan-Chia Ou ◽  
Jui-Hung Shien ◽  
Poa-Chun Chang
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Virus Type ◽  
Vaccine Strain ◽  
Hepatitis A ◽  
High Resolution Melting ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Duck Hepatitis

Duck hepatitis A virus type 1 (DHAV-1) infection is a highly contagious and fatal disease of young ducklings. A live attenuated vaccine strain designated as 5886 has been used in Taiwan for the control of DHAV-1. Although several molecular biological methods are reported for diagnosis of DHAV-1 infection, none of them is able to discriminate between the vaccine strain and field viruses of DHAV-1. In the present study, a real-time reverse transcriptase polymerase chain reaction (RT-PCR) and high resolution melting (HRM) assay was developed for rapid detection and differentiation between the vaccine strain and field viruses of DHAV-1. This assay is highly specific for DHAV-1 and the detection limit is about 100 copies of the viral RNA. Experiments using fecal samples collected from ducklings experimentally infected with DHAV-1 showed that DHAV-1 could be detected in fecal samples as early as 6 h post-infection. In summary, a real-time RT-PCR and HRM assay is developed in this study and this assay could be valuable for diagnosis and surveillance of DHAV-1 infection in the field.

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