Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus

Abstract

Introduction: The hepatitis B virus (HBV), HCV, and HEV may occur as singly or concurrently in patients of different kind of liver disease. The rapid, reliable, and cost-effective screening of these pathogens is required for the large epidemiological studies. Therefore, a study has been planned to develop a multiplex Reverse Transcriptase-PCR assay which can be used for the screening of maximum number of pathogens at a time. Methodology: To develop multiplex Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay for simultaneous detection of HBV, HCV, and HEV; the serum samples of 54 patients who were positive either singly or in co-infection with for HBV, HCV, and HEV serologically were screened by uniplex PCR/RT-PCR followed by multiplex RT-PCR for HBV, HCV, and HEV using specific primers. These primers can detect most genotypes of these viruses. Multiplex RT-PCR was done in one tube for the identification of viral DNA/RNA using a mixture of three pairs of specific primers for hepatitis B, C, and E viruses. Representative positive samples of these viruses by uniplex/multiplex RT-PCR were also confirmed by sequencing followed by alignment with reference strains sequence. Results: The specificity of multiplex PCR was 100% with high sensitivity 89%, 87%, and 74% for HBV, HCV, and HEV respectively. The sensitivity and specificity of RT-multiplex PCR demonstrated a good correlation with that of uniplex PCR. Conclusion: The study suggests that multiplex RT-PCR can serve as a simple and reliable assay for rapid, sensitive, and cost-effective method for simultaneous detection of super-infections with HEV particularly in Asian countries as a cause of decompensation of chronic liver disease. ( J CLIN EXP HEPATOL 2016;6:33-39)

NO1, a New Sigma 2 Receptor/TMEM97 Fluorescent Ligand, Downregulates SOCE and Promotes Apoptosis in the Triple Negative Breast Cancer Cell Lines

“…The inhibitory effect of NO1 on SOCE was consistent with its effect impairing STIM1/Orai1 interaction, and probably, by promoting the dissociation of the complex between σ2R/TMEM97 and STIM1. As a result, NO1 administration to MDA-MB-231 cells evoked a reduction in cell proliferation and migration that agrees with previous investigations reported in the literature using other ligands of σ2R/TMEM97 [17,18,36,61]. In line with our observations, σ2R/TMEM97 was shown to be overexpressed in different cancer types, and even in patients with breast cancer [31,62,63].…”

Virological and Immunological Outcomes of Coinfections

“…However, PCR is labor-intensive and expensive, particularly for the detection of multiple viral agents. Quantitative real-time PCR (qRT-PCR) for concurrent detection of heterogeneous viruses in a single reaction has reduced the overall cost (66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81). Although the qRT-PCR system is quite sensitive, adsorption and fluorescence spectra for different fluorophores used in the fluorescent-labeled probe systems tend to interfere with each other, which limits reliable detection to a maximum 4 or 5 different viruses (82).…”

Economic evaluation of implementing a decentralised Hepatitis B virus diagnostic intervention under National Viral Hepatitis Control Programme in Tamil Nadu, South India

“…All costs were calculated in Indian rupees () and converted to USD ($) according to the 2019 exchange rate of $1=69. Sensitivity and specificity of rapid test and ELISA were collected from relevant published studies (Table 2) [23,24,25,26,27]. For prevalence of HBV, we conducted systematic review from published prevalence studies done in Tamil Nadu and was used to populate the model (Table 3).…”