A SNaPshot assay for the rapid and simple detection of hepatitis B virus genotypes

Abstract

A simple technique for the identification of common genotypes of the hepatitis B virus (HBV) remains to be identified. The present study was conducted to establish such a methodology. Four plasmids of genotypes A-D and 123 clinical serum specimens of HBV-infected patients were genotyped. HBV genotypes would be detected successfully when the HBV genotype reached a viral load of 1 × 103 copies/ml or the BC genotype mixed samples reached a 5% level. The lower limit of detection of HBV DNA in serum specimens was determined to be 2.14×102 IU/ml. The assay sensitivity and specificity were 100% and the consistency was demonstrated to reach as high as 90.24 and 100% compared with that of the DNA sequencing and cloning. The frequencies of the genotypes B, C, BC, BD and BCD were found to be 65.0, 23.6, 7.3, 3.3 and 0.8%, respectively. The accuracy of detection of the mixed infections was also higher using the rapid and simple SNaPshot method compared with that achieved with the DNA sequencing methods. The results of the present study indicated that the SNaPshot technique accurately distinguishes the HBV genotypes A-D and is able to be readily applied as a monitoring tool in HBV prognosis and treatment.

Correlation Between Pneumocystis jirovecii Mitochondrial Genotypes and High and Low Fungal Loads Assessed by Single Nucleotide Primer Extension Assay and Quantitative Real‐Time PCR

“…As the sensitivity threshold of the present SNaPshot method assay is between 5% and 10% as shown by the reproducibility study and in accordance with previous reports of this method (Lai et al. ), this percentage may be seen as a probable minimum. Using a similar SNaPshot method, Esteves et al.…”

“…In this study of a single locus (mt85 SNP of the mt LSU rRNA gene), we found variability to be high, with 27% (36/133) of samples containing multiple alleles. As the sensitivity threshold of the present SNaPshot method assay is between 5% and 10% as shown by the reproducibility study and in accordance with previous reports of this method (Lai et al 2014), this percentage may be seen as a probable minimum. Using a similar SNaPshot method, Esteves et al (2011)reported a lower frequency of mixtures, at 19%, for tests on 31 samples.…”

“…SNaPshot can detect minority alleles with frequencies as low as 5–10% (Lai et al. ) and is therefore adapted for detecting mixtures of alleles. In parallel, we used quantitative PCR (qPCR) to determine the fungal load in the bronchoalveolar (BAL) fluid samples tested (Botterel et al.…”

“…As Sanger sequencing minority allele detection is reliable only for alleles with a frequency of more than 30% (Angulo et al 2010), we designed a single nucleotide primer extension (SNaPshot) assay. SNaPshot can detect minority alleles with frequencies as low as 5-10% (Lai et al 2014) and is therefore adapted for detecting mixtures of alleles. In parallel, we used quantitative PCR (qPCR) to determine the fungal load in the bronchoalveolar (BAL) fluid samples tested (Botterel et al 2012).…”

A multiplex method for detection of glucose‐6‐phosphate dehydrogenase (G6PD) gene mutations

“…With this approach, multiplex extension primers with different lengths and labeled with four fluorescently labeled dNTPs (A, T, G, and C) of four colors (green, red, blue, and black) allow the detection of different single nucleotide polymorphisms (SNPs) shown as single-peak fluorescence wave forms in an electropherogram on an automated sequencer. It has been utilized to screen SNPs with high accuracy and effectiveness and enables rapid detection of mutations in both homozygotes and heterozygotes [26][27][28][29][30][31].…”