Broadly Reactive Real-Time RT-PCR Assay for the Detection of Hepatitis E Virus and Simultaneous Genotyping by Single Nucleotide Polymorphism Analysis

Ishida, Setsuko; Yoshizumi, Shima; Sakata, Hiromi; Matsubayashi, Keiji

doi:10.1128/spectrum.01912-21

Cited by 4 publications

(5 citation statements)

References 57 publications

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“…However, PCR is usually combined with other techniques (such as PCR sequencing, PCR-RFLP, PCR-fluorescent probe method, and PCR melting analysis) to analyze similar targets. Among these techniques, the PCR-fluorescent probe method is the most commonly used in clinical settings for polymorphism analysis. ,, However, there are still some drawbacks that hindered its usage in mutipolymorphism site detection. For example, the PCR-fluorescent probe method relies on strict annealing temperature control for high annealing temperature, which leads to the departure of all probes from the target, and low annealing temperature leads to probes binding without selection.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Yao

Zhang

2022

Anal. Chem.

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Genotyping of folate metabolism genes is of great importance in disease diagnosis and prevention. However, most current detection methods used for folate metabolism gene genotyping are based on sequencing and chips, which suffer from a high cost and laborious and time-consuming procedures. Herein, we reported a multiplex asymmetric PCR-HRM strategy for identifying genotypes of folate metabolism genes in a single tube. The proposed multiplex PCR-HRM assay has been successfully applied to identify the genotypes of folate metabolism genes, methylene tetrahydrofolate reductase (C677T, A1298C) and methionine synthase reductase A66G, on 1 μL of genomic DNA (gDNA) samples directly released from blood specimens, and the genotyping results were 100% consistent with the results of sequencing. The assay allows us to accurately detect the genotypes of gDNA with the detection limit down to 1 ng, which meets the clinical requirement. What is more, the capacity of resistance to aerosol pollution of the multiplex asymmetric PCR-HRM biosensing was first addressed and has been evaluated as it can withstand contamination of roughly 12.5–25% interfering nucleic acids. Because of the advantages of multiplex detection, high accuracy, and resistance to aerosol pollution and having no open tube procedure, this approach would pave the way for establishing a fast and cost-effective platform for folate metabolism gene genotyping and other SNP genotyping in clinical diagnostics.

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Section: Introductionmentioning

confidence: 99%

Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Yao

Zhang

2022

Anal. Chem.

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“…HEV genotypes 1 and 2 are clearly responsible for human hepatitis, and they are spread through the fecal-oral route via contaminated water in Asia and Africa and in Africa and Mexico, respectively [7]. The incidence of autochthonous HEV infections (predominantly genotypes 3 and 4) is increasing in industrialized countries such as Japan [25][26][27]. The present case supports the previous reports that HEV genotype 3, as well as other genotypes, can induce acute-on-chronic liver failure (ACLF) in patients with underlying liver diseases [28][29][30].…”

Section: Discussionmentioning

confidence: 99%

Hepatitis E Virus Infection Caused Elevation of Alanine Aminotransferase Levels in a Patient with Chronic Hepatitis B and Choledocholithiasis

Hirano,

Kanda,

Honda

et al. 2023

Reports

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Hepatitis E virus (HEV) genotypes 3 and 4 are zoonotic strains that are primarily transmitted through the consumption of undercooked pork or game meat. They also cause asymptomatic infections, acute hepatitis, acute-on-chronic liver failure, chronic hepatitis, and extrahepatic manifestations. Here, we report a man in his 80s who had chronic hepatitis B, took entecavir for it, and presented with higher levels of alanine aminotransferase (ALT) and jaundice. An abdominal computed tomography scan revealed choledocholithiasis with cholecystolithiasis. Although endoscopic papillary balloon dilatation was performed for the removal of a common bile duct stone, the abnormal liver function tests, including jaundice, were prolonged. After other viral hepatitis and other causes of the liver injury were ruled out, as his serum was positive for immunoglobulin A anti-HEV and HEV genotype 3b RNA, we diagnosed him as having acute hepatitis E. In this case, with chronic hepatitis B and a common bile duct stone, the prolonged abnormal results for the liver function tests seemed to be caused by HEV infection. In conclusion, in cases with high ALT levels after removing choledocholithiasis, other factors, including HEV infection, should be considered to determine the cause of abnormal liver function test results. The further examination of hepatitis D virus infection and high ALT levels may be needed in HBV-infected individuals.

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“…The protein and RNA of HEV are considered to be infectivity markers. To date, several conventional RT‐PCR, real‐time quantitative PCR (qPCR), and loop‐mediated isothermal amplification methods have been developed for the detection of HEV RNA in serum and plasma or fecal samples 105‐107 . qPCR for detecting HEV RNA has become the gold standard for the diagnosis of HEV infection in humans and animals 107 .…”

Section: Clinical Significance Of the Hev Orf3 Protein As A Diagnosti...mentioning

confidence: 99%

“…To date, several conventional RT-PCR, real-time quantitative PCR (qPCR), and loop-mediated isothermal amplification methods have been developed for the detection of HEV RNA in serum and plasma or fecal samples. [105][106][107] qPCR for detecting HEV RNA has become the gold standard for the diagnosis of HEV infection in humans and animals. 107 Detection of HEV proteins mostly targets the ORF2 protein, 21,108,109 but its detection has problems such as quasi-enveloped shielding antigen and glycosylated ORF2 secreted by dimers not associated with virions, which leads to the inability to determine the actual viral load of the patient.…”

Section: Clinical Significance Of the Hev Orf3 Protein As A Diagnosti...mentioning

confidence: 99%

Multifunctional ORF3 protein of hepatitis E virus

Li,

Sun,

Pinpin

et al. 2024

Journal of Medical Virology

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Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is transmitted primarily through the fecal‐oral route and can cause acute hepatitis in humans. Since HEV was identified as a zoonotic pathogen, different species of HEV strains have been globally identified from various hosts, leading to an expanding range of hosts. The HEV genome consists of a 5' noncoding region, three open reading frames (ORFs), and a 3' noncoding region. The ORF3 protein is the smallest but has many functions in HEV release and pathogenesis. In this review, we systematically summarize recent progress in understanding the functions of the HEV ORF3 protein in virion release, biogenesis of quasi‐enveloped viruses, antigenicity, and host environmental regulation. This review will help us to understand HEV replication and pathogenesis mechanisms better.

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Broadly Reactive Real-Time RT-PCR Assay for the Detection of Hepatitis E Virus and Simultaneous Genotyping by Single Nucleotide Polymorphism Analysis

Abstract: The distribution of HEV-3 and HEV-4 has been changing. HEV-4, which had been predominantly found in Asia, is now being detected in other parts of the world, and there are now reports of chronic infections.

Cited by 4 publications

References 57 publications

Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Hepatitis E Virus Infection Caused Elevation of Alanine Aminotransferase Levels in a Patient with Chronic Hepatitis B and Choledocholithiasis

Multifunctional ORF3 protein of hepatitis E virus

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