单管多重荧光定量RT-PCR方法鉴别A组轮状病毒及诺如病毒GⅠ型和GⅡ型  

Discrimination of rotavirus and NoV GⅠ and NoV GⅡ using a single- tube multiplex real- time RT- PCR

作　　者：曹芳芳[1] 徐德顺[2] 

机构地区：[1]湖州市中医院,浙江湖州313000 [2]湖州市疾病预防控制中心,浙江湖州313000

出　　处：《中国卫生检验杂志》2015年第6期823-826,共4页Chinese Journal of Health Laboratory Technology

摘　　要：目的建立能同时检测A组轮状病毒、诺如病毒GⅠ型和GⅡ型的多重荧光定量RT-PCR方法。方法分别选用轮状病毒NSP3基因、诺如病毒GⅠ型和GⅡ型orf1与orf2基因中的64 bp、90 bp和185 bp片段作为荧光定量PCR检测的靶标,设计引物和Taq Man探针,构建标准品,绘制标准曲线,评价检测方法的重复性、特异度和灵敏度,并对138份粪便标本进行检测及测序验证。结果 3种病毒对应的标准曲线分别为:轮状病毒:y=-3.9lgx+41.204;诺如病毒GⅠ型:y=-3.06lgx+40.716;诺如病毒GⅡ型:y=-3.29lgx+41.538,质粒标准曲线的方差系数为0.997,灵敏度达到每个反应102copy/μl,特异度强。138份样本中检出A组轮状病毒、诺如病毒GⅠ型和GⅡ型的检出率分别为32.6%、5.8%和26.1%,与基因测序比对结果相符。结论构建可用于A组轮状病毒及诺如病毒GⅠ型和GⅡ型的多重荧光定量RT-PCR方法快速、特异且灵敏,可用于临床病原诊断和流行病学调查。Objective To establish a single- tube multiplex real- time RT- PCR assay for detecting rotavirus and No V GⅠ and No V GⅡ simultaneously. Methods 64 bp fragment in NSP3 gene of rotavirus group A,90 bp fragment in orf1 and orf2 gene of No V GⅠ and 185 bp fragment in orf1 and orf2 gene of No V GⅡ were chosen as the target genes for multiplex real-time RT- PCR,a specific primers and probes labeled with different fluoresceins were designed. The standard plasmid was constructed,and the standard curve was established. The reproducibility,specificity and sensitivity of the assay were evaluated.Furthermore,RNA extracted from 138 fecal samples of patients with viral diarrhea was detected and verified by sequencing. Results The corresponding standard curves of rotavirus,No V GⅠ and No V GⅡ were y =- 3. 9lgx ＋ 41. 204,y =- 3. 06 lgx ＋40. 716,y =- 3. 29 lgx ＋ 41. 538,respectively; The standard curve coefficient was 0. 997. The detection limit of this assay was102 copy / μl in one reaction,the specificity was severe. The detection rates of 138 fecal samples were respectively 32. 6%,5. 8% and 26. 1% for rotavirus,No V GⅠ and No V GⅡ RNA,which was coincident with the sequencing results. Conclusion The single- tube multiplex real- time RT- PCR assay developed in this study for detecting and identifying rotavirus,No V GⅠ and No V GⅡ was rapid,specific and sensitive. It can be used for clinical diagnosis and epidemiological investigation on pathogen.

关 键 词：轮状病毒 诺如病毒 多重荧光逆转录-聚合酶链反应 GⅠ型 GⅡ型 

分 类 号：R446.5[医药卫生—诊断学]