四种食源性病毒多重反转录-聚合酶链反应检测研究

寇晓霞; 吴清平; 姚琳; 张菊梅

Abstract

寇晓霞,吴清平,姚琳,张菊梅.四种食源性病毒多重反转录-聚合酶链反应检测研究[J].Chinese journal of Epidemiology,2008,29(6):590-593

四种食源性病毒多重反转录-聚合酶链反应检测研究

Studies on simultaneous detection of four foodborneviruses by multiplex reverse transcription-polymerase chain reaction

Received:October 11, 2007

DOI：10.3321/j.issn:0254-6450.2008.06.018

KeyWord: 食源性病毒|多重反转录-聚合酶链反应|检测

English Key Word: Foodbornevirus|Multiplex reverse transcription-polymerase chain reaction|Detection

FundProject:广东省科技计划资助项目(2007AD50100001)

Author Name	Affiliation	E-mail
KOU Xiao-xia	Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Instituteof Microbiology, Guangdong 510070, China
WU Qing-ping	Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Instituteof Microbiology, Guangdong 510070, China	wuqp203@yahoo．com．cn
YAO Lin	中国科学院武汉病毒研究所
ZHANG Ju-mei	Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Instituteof Microbiology, Guangdong 510070, China

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Abstract:

目的建立同时检测诺如病毒、轮状病毒、星状病毒和甲肝病毒多重RT-PCR检测方法．方法以四种食源性病毒的高度保守区为靶序列设计特异引物，优化反应体系和条件，确定多重RT-PCR检测四种病毒的特异性和灵敏度，并初步应用于临床样本中四种病毒的同时检测．结果在灵敏度试验中得到的轮状病毒、诺如病毒和星状病毒稳定的最高检测限均为50 pg/ml，甲肝病毒为100 pg/ml．在128份临床粪便样本中，其中轮状病毒阳性62份(48.44%)，诺如病毒阳性8份(6.25%)，星状病毒阳性11份(8.59%)，甲肝病毒阳性4份(3.12%)．结论研究所建立的多重RT-PCR方法，在实际应用中能同时处理大量的样本，提高PCR检测方法的能力，可以应用于临床病例的诊断和流行病学调查等研究．

English Abstract:

Objective To establish a method for simultaneous detection of norovirus (NV),rotavirus (RV), astrovirus (AV) and hepatitis A virus (HAV) by multiplex reverse transcriptionpolymerase chain reaction (RT-PCR). Methods Specific primers of the four viruses were designed based on the high conserved sequences, the reaction system and conditions optimized and the specificity and sensitivity confirmed. The method was then applied to detect the four viruses in clinical samples. Results The steady detection limits were 100 pg/ml for hepatitis A virus, 50 pg/ml for rotavirus, norovirus and astrovirus respectively. When the developed method was used to detect clinical fecal samples, 62(48.44%)were iden tified as rotavirus, 8 (6.25%) as norovirus, 11 (8.59%) as astrovirus and 4 (3.12%) as hepatitis A virus in a total of 128 samples. Conclusion Data from our study showed that multiplex RTPCR system could be used to simultaneously detect the four viruses in routine monitoring and risk assessment in disease outbreaks with high specificity and sensitivity.

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