诺如病毒遗传组Ⅱ型TaqMan—MGB探针实时荧光PCR的检测研究 |
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引用本文: | 陈华,谭翰清,郭赐贶,谭海芳,程洁萍,林凤,孔秀珍,丁丽娜.诺如病毒遗传组Ⅱ型TaqMan—MGB探针实时荧光PCR的检测研究[J].广东卫生防疫,2008(1):22-25. |
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作者姓名: | 陈华 谭翰清 郭赐贶 谭海芳 程洁萍 林凤 孔秀珍 丁丽娜 |
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作者单位: | 肇庆市疾病预防控制中心,广东肇庆526020 |
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基金项目: | 肇庆市科技计划项目(2006E1818) |
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摘 要: | 目的建立特异、灵敏的诺如病毒遗传组Ⅱ型TaqMan—MGB探针实时荧光PCR快速检测方法。方法根据GenBank诺如病毒遗传组Ⅱ型代表株保守序列设计特异引物对和TaqMan~MGB探针,调整引物、探针浓度,优化最佳反应条件,建立一步法实时荧光PCR快速检测反应体系,进行灵敏度、特异性、稳定性试验,以评价反应体系,并与引物P289/P290常规RT—PCR进行灵敏度比较。结果诺如病毒遗传组Ⅱ型TaqMan—MGB探针实时荧光PCR检测时限短,仅1h就出结果;最低检出下限为100拷贝/mL,比引物P289/P290常规RT—PCR灵敏度高100倍;与轮状病毒、腺病毒、星状病毒、甲肝病毒无交叉反应;4份核酸含量不同的标本重复检测5次,平均G值变异系数范围0.96%-2.27%。结论诺如病毒遗传组Ⅱ型TaqMan—MGB探针实时荧光PCR快速、特异、灵敏、稳定性好。可应用于突发公共卫生应急检测,提高快速检测能力。
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关 键 词: | 人类杯状病毒 诺如病毒 实时荧光PCR 逆转录PCR. TaqMan MGB探针 |
Detection of genotype H of norovirus by TaqMan-MGB probe-based real-time fluorescent polymerase chain reaction |
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Institution: | CHEN Hua, TAN Han-qing, GUO Ci-kuang, et al.( Zhaoqing Center for Disease Control and Prevention , Zhaoqing 526020, China ) |
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Abstract: | Objective To establish a rapid, specific and sensitive molecular method to detect genotype Ⅱ of norovlrus using TaqMan-MGB probe-based real-time fluorescent polymerase chain reaction. Methods Specific primers and TaqMan-MGB probe were designed according to genotype Ⅱ conserved sequence of norovirus from GenBank, and then optimized concentration of primers and probe. Reaction condition was introduced to establish one step TaqMan-MGB probe-based real-time PCR system and compared the conventional RT-PCR with P 289/P 290 primers to evaluate specificity, sensitivity and stability. Results The one step TaqMan-MGB probe-based real-time PCR method found rapid and sensitive with 1 hour time limit and 100 copies/mL lowest detection limit, No cross reaction observed with other viruses including rotavirus, adenovirus, astovirus and hepatitis A virus. The coefficients of variation of the average C1 values ranged 0.96%-2.27% in 4 different virus nuclei acid concentration samples after tested repeatedly for 5 times. Conclusion The one step TaqMan-MGB probe-based real-time PCR method was rapid, specific, sensitive and stable to detect genotype Ⅱ of norovirus and had potential application in emergent response. |
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Keywords: | Human calicivirus Norovirus Real-time PCR RT-PCR TaqMan MGB probe |
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