Xie Zhixun, Xie Liji, Pang Yaoshan, Lu Zhaofa, Xie Zhiqin, Sun Jianhua, Deng Xianwen, Liu Jiabo, Tang Xiaofei, Khan Mazhar
Department of Biotechnology, Guangxi Veterinary Research Institute, 51 You Ai Road, 530001, Nanning, Guangxi, China.
Arch Virol. 2008;153(12):2245-51. doi: 10.1007/s00705-008-0253-0. Epub 2008 Nov 19.
A real-time multiplex polymerase chain reaction (rtm-PCR) assay was developed and optimized to simultaneously detect three viral pathogens of shrimp in one reaction. Three sets of specific oligonucleotide primers for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV), along with three TaqMan probes specific for each virus were used in the assay. The rtm-PCR results were detected and analyzed using the Light Cycler 2.0 system. Forty-five PCR-positive samples and four negative samples were used to confirm the sensitivity and specificity of the rtm-PCR. The rtm-PCR identified and differentiated the three pathogens. With one viral infection of shrimp, a specific amplified standard curve was displayed. When samples from shrimp infected with two or three pathogens were analyzed, two or three specific standard curves were displayed. The sensitivity of the rtm-PCR assay was 2,000, 20, and 2,000 template copies for WSSV, IHHNV and TSV, respectively. No positive results (standard curves) were displayed when nucleic acid from Vibro spp., and Streptococcus spp. DNA were used as PCR templates. The results indicate that real-time multiplex PCR is able to detect the presence of and differentiate each pathogen in infected shrimp. This real-time multiplex PCR assay is a quick, sensitive, and specific test for detection of WSSV, IHHNV and TSV and will be useful for the control of these viruses in shrimp.
开发并优化了一种实时多重聚合酶链反应（rtm-PCR）检测方法，以在一次反应中同时检测虾的三种病毒病原体。该检测方法使用了针对白斑综合征病毒（WSSV）、传染性皮下和造血组织坏死病毒（IHHNV）以及桃拉综合征病毒（TSV）的三组特异性寡核苷酸引物，以及针对每种病毒的三种TaqMan探针。使用Light Cycler 2.0系统检测和分析rtm-PCR结果。使用45个PCR阳性样本和4个阴性样本确认rtm-PCR的灵敏度和特异性。rtm-PCR能够识别和区分这三种病原体。对于感染一种病毒的虾，会显示出一条特异性扩增标准曲线。当分析感染两种或三种病原体的虾的样本时，会显示出两条或三条特异性标准曲线。rtm-PCR检测方法对WSSV、IHHNV和TSV的灵敏度分别为2000、20和2000个模板拷贝。当使用弧菌属和链球菌属DNA的核酸作为PCR模板时，未显示出阳性结果（标准曲线）。结果表明，实时多重PCR能够检测感染虾中每种病原体的存在并进行区分。这种实时多重PCR检测方法是一种快速、灵敏且特异的检测WSSV、IHHNV和TSV的方法，将有助于控制虾中的这些病毒。
