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As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

Background: Polymorphism in genes associated with Vitamin-D metabolism and its receptor molecule altogether modulates the level of active serum Vitamin-D level and as such serves as potent immune modulator. Impact of this polymorphism on outcome of HBV related liver disease is poorly understood till date. Aim: This study focus on analysing the VDR polymorphism (TaqI, ApaI, and BsmI) and its associated molecules GC-Globulin and CYP2R1 among HBV infected patients and its probable correlation with disease progression. Method: Three hundred forty HBV infected patients belonging to three distinct clinical groups, Acute Viral hepatitis (AVH, n=205), Chronic Hepatitis (CH, n=84) and hepatocellular carcinoma (HCC, n = 51) along with 102 healthy control were included in the study. VDR, GC and CYP2R1 polymorphism were genotyped by PCR-RFLP method followed by Sanger sequencing. Haplotype distribution was analysed using SHEsis software. Logistic regression analysis was performed to find the association between different genotype and VDR haplotype with progression of liver disease. Further, an SVM based prediction model has also been described in detection of different disease stage. Results and Conclusion: Apa-I CC genotype was more frequently observed among chronic HBV patients and HCC than the acute cases among North east Indian population (OR = 2.241, C.I = 0.504- 5.558. p= 0.001). The occurrence of bAt haplotype was also found to be significantly higher among Chronic (p=0.004) and HCC (p=0.001<0.05) cases in comparison to acute cases. Further, logistic regression analysis after adjusting covariates like age, gender, serum AST/ALT level, serum albumin and platelet count, showed that Apa-I CC genotype and bAt haplotype were independent predictors for advancement of acute to chronic HBV infection and further. The SVM based prediction model using similar covariates also predicts the different disease stage with 90% accuracy.

Background:With the increasing COVID-19 infection worldwide, economization of the existing RT-PCR based detection assay becomes the need of the hour. Methods: An assessment of optimal PCR conditions for simultaneous amplification for E, S and RdRp gene of SARS-CoV-2 has been made using both fast traditional and multiplex real time PCR using same primer sets. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19. Results: The designed primers for amplification of E, S and RdRp gene of SARS-Cov-2 in single tube Multiplex PCR amplifications have shown efficient amplification of the target region in 37 minutes using thermal cyclers and 169 minutes with HRM based Real time detection using SYBR green master mix, over a wide range of template concentration, and the results were in good concordance with the commercially available detection kits. Conclusion: This fast HRM based Real time multiplex PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost effective manner apart from the added advantage of primer pair’s compatibility for use in Traditional multiplex PCR, which offers extended applicability of the assay protocol in resource limited setting.

AIMS: To study the genetic association of cardiac conduction defects (CCD) by evaluating Single nucleotide polymorphism(SNP) in genes of SCN1B and KCNJ2 and to evaluate baseline characteristics between cases and controls. METHODS AND RESULTS: Case group consisted of 81 individuals with diagnosis of conduction disturbances who underwent permanent pacemaker implantation. . The control group consisted of 79 unrelated individuals above 18 years of age of the local population not having a present or past personal or family history (first degree relatives) of any cardiac ailment especially cardiac conduction defects. Isolation of genomic Deoxyribonucleic acid(DNA) from all samples was done, Genomic DNA was checked to ensure the presence of intact DNA . SCN1B : SNP rs55742440 have no bearing on the protein except in producing a splice variant . SNP rs67701503 does not lie in the splice site region, not having any significance in the regulation of the gene as well. NetGene2 analysis of SNP rs67486287 negates its presence in the splice site. KCNJ2 :SNP rs199473653 is leading to a missense amino acid change resulting in homozygous GG variant found in almost equal frequency in both groups. SNP rs199473653 gene has not been reported as a disease-causing mutation. CONCLUSION:The alteration of nucleotide in SCN1B intron (SNP rs55742440, rs67701503, rs 67486287) between cases and controls was found to have no odds of affecting the outcome of CCD. There was no variation or alteration in nucleotide bases of KCNJ2 (SNP rs786205813, rs199473653) between the groups.