蜜蜂KBV和APV病毒RT-PCR检测技术研究

Study on Detection of Kashmier Bee Virus and Acute Paralysis Virus by RT-PCR in Honeybees

根据蜜蜂克什米尔病毒(Kashmirbeevirus,KBV)和急性麻痹病毒(Acuteparalysisvirus,APV)的RNA聚合酶基因序列,参照DonStoltz报道的引物KBV1、KBV2,美国农业部提供的引物DC62F、DC682R,对美国农业部惠赠的标准KBV和APV的RNA聚合酶基因片段,以及2001-2002年收集到的中国21个省市的180份疑似KBV和APV病蜂RNA聚合酶基因片段进行RT-PCR。对以KBV1、KBV2和DC62F、DC682R为引物扩增出的RNA聚合酶基因片段进行克隆、转化,并对克隆结果测序,证明其可靠性。建立了用分子生物学方法检测蜜蜂KBV和APV病毒的技术,有一定的应用前景。检测结果表明:到目前为止,我国蜜蜂群中未发现KBV和APV病毒病。

The detection of kashmir bee virus(KBV) and acute paralysis virus(APV) was conducted in honeybee. By using RNA polymerase gene sequences of KBV and APV and proper primers, RT-PCR was developed on standard KBV and APV RNA polymerase gene fragments, and honeybee samples collected in 21 different provinces around China during the period of 2001-2002,which were suspected infected KBV or APV. A pair of primers (KBV1, KBV2) for KBV amplification was designed according to the report of Don Stoltz. The other set of primers (DC62F, DC682R) for APV and KBV, standard KBV and APV RNA polymerase gene fragments were all given by United States Department of Agriculture. A 414 bp amplicon from standard KBV polymerase gene fragments was amplified using KBV1, KBV2 and a 602bp amplicon was amplified using DC62F DC682R from standard KBV and APV polymerase gene fragments. At the same time the products acquired were cloned and sequenced. The results showed that no KBV and APV of honeybee were found in China up to now and the method established in this paper is specific, sensitive and simple for detection and diagnosis of KBV and APV diseases.