Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis

• 作者: Bonita E Lee Xiaoli L Pang Yupin Tong
• 来源期刊: 世界病毒学杂志
• 学科: 医学
• 关键词: ROTAVIRUS A Melting temperature REAL-TIME POLYMERASE chain reaction SYBR green GENOTYPING
• 年，卷（期）: 2015,（4）
• 页码范围: 365-371
• 页数: 7页
• 分类号: R

摘要

AIM:To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR)assay to genotype rotavirus(G and P)in Alberta from January 2012 to June 2013.METHODS:We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-gPCR)by selecting genotype-specific primers of published conventional RT nested PCR(cnRT-PCR)assay and optimizing the amplification conditions.cDNA was first synthesized from total RNA with SuperScript?Ⅱreverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR.After the PCR reaction,melting curve analysis was used to determine specific genotype.Sixteen samples previously genotyped using cnRT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis.Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay.The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method.After validation and optimization,the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January RESULTS:The new rt-gPCR assay was validated and optimized.The assay detected G1 to G4,G9,G12 and P[4]and P[8]that were available as positive controls in our laboratory.A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80℃to 82℃.The sensitivity of rt-gPCR was comparable to cnRT-PCR with 100%correlation of the 16 samples with known G and P genotypes.No cross reaction was found with other gastroenteritis viruses.Using the new rt-gPCR assay,genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus:G1P[8](42.6%),G2P[4](4.9%),G3P[8](10.7%),G9P[8](10.7%),G9P[4](6.6%),G12P[8](23.0%),and unknown GP[8](0.8%).For the first time,G12 rotavirus strains were found in Alberta and G12 was