蜜蜂杂志
蜜蜂雜誌
밀봉잡지
JOURNAL OF BEE
2012年
4期
3-6
,共4页
蜜蜂畸翅病毒%荧光定量%RT—PCR%特异性%灵敏度
蜜蜂畸翅病毒%熒光定量%RT—PCR%特異性%靈敏度
밀봉기시병독%형광정량%RT—PCR%특이성%령민도
Deformed Wing Virus%real time quantitativeRT-PCR%Kunming%infect frequencies
为建立蜜蜂畸翅病毒SYBR Green Ⅰ荧光定量RT—PCR检测方法，通过构建蜜蜂畸翅病毒（DWV）RDRP基因部分序列的标准质粒作为模板，进行了标准曲线的建立，并对所建立方法进行了特异性和灵敏度测试。结果表明：所建立的荧光定量RT—PCR扩增效率E=100％，R^2=0．999，最低检出量为100个拷贝；该方法仅能检出DWV病毒，而不能检测出蜜蜂黑王台病毒、囊状幼虫病毒，具有很好的特异性。表明已成功建立了特异灵敏的DWV实时荧光定量RT—PCR检测方法。采用所建方法对昆明部分蜂场进行了DWV感染状况调查，结果表明昆明地区东方蜜蜂带毒率为54％，提示DWV病毒在昆明地区东方蜜蜂中存在流行。
為建立蜜蜂畸翅病毒SYBR Green Ⅰ熒光定量RT—PCR檢測方法，通過構建蜜蜂畸翅病毒（DWV）RDRP基因部分序列的標準質粒作為模闆，進行瞭標準麯線的建立，併對所建立方法進行瞭特異性和靈敏度測試。結果錶明：所建立的熒光定量RT—PCR擴增效率E=100％，R^2=0．999，最低檢齣量為100箇拷貝；該方法僅能檢齣DWV病毒，而不能檢測齣蜜蜂黑王檯病毒、囊狀幼蟲病毒，具有很好的特異性。錶明已成功建立瞭特異靈敏的DWV實時熒光定量RT—PCR檢測方法。採用所建方法對昆明部分蜂場進行瞭DWV感染狀況調查，結果錶明昆明地區東方蜜蜂帶毒率為54％，提示DWV病毒在昆明地區東方蜜蜂中存在流行。
위건립밀봉기시병독SYBR Green Ⅰ형광정량RT—PCR검측방법，통과구건밀봉기시병독（DWV）RDRP기인부분서렬적표준질립작위모판，진행료표준곡선적건립，병대소건립방법진행료특이성화령민도측시。결과표명：소건립적형광정량RT—PCR확증효솔E=100％，R^2=0．999，최저검출량위100개고패；해방법부능검출DWV병독，이불능검측출밀봉흑왕태병독、낭상유충병독，구유흔호적특이성。표명이성공건립료특이령민적DWV실시형광정량RT—PCR검측방법。채용소건방법대곤명부분봉장진행료DWV감염상황조사，결과표명곤명지구동방밀봉대독솔위54％，제시DWV병독재곤명지구동방밀봉중존재류행。
Area l-time RT-PCR assay, based on SYBR Green （SG） chemistry, were developed, for the detection of a important honeybee viruse： Deformed Wing Virus （DWV）, the reaction were optimized to yield the highest sensitivity and specificity. Viral detection and identification were confirmed by melting curve analysis and sequencing of the PCR products. The R2 was 0.999 and the effciency of the PCR reaction was 100%. The detection limit for the DWV RT-PCR was determined by the lowest dilution that showed specifc amplification for the positive control, which was 102 copies. The technique was used to evaluate Kunming feld samples and establish the distribution of the viruse. Sacbrood virus was present at 54% frequencies.
