柯萨奇B组病毒逆转录聚合酶链反应检测方法的建立

Detection of Coxsackievirus B Group RNA by Reversetranscription Polymerase Chain Reaction

根据柯萨奇Ｂ组病毒（ＣＢＶ）基因组５’非编码区核苷酸序列，选用了一对组特异性引物，用于逆转录聚合酶链反应检测ＣＢＶ－ＲＮＡ。结果显示该法可检出ＣＢＶ１～６型ＲＮＡ，灵敏度为１０ＰＦＵ，不能检出其他病毒ＲＮＡ。采用的碘化钠－异硫氰酸胍－氯仿法提取ＣＢＶ－ＲＮＡ与传统的蛋白酶Ｋ－酚－氯仿法及异硫氰酸胍－酚－氯仿法比较，具有快速、简便、稳定、可靠的特点。应用该法检测ＣＢＶ－５感染鼠外周血ＲＮＡ，第３ｄ即为阳性。

By taking advantage of highly conserved 5’ nocoding region of the coxsackievirus B group (CBV) genome. We designed a pair of CBV group specific primers. The method of reversetranscription polymerase chain reaction(RT PCR) was developed to detect CBV RNA. The results showed that CBV of 1 to 6 types could be detected by RT PCR, the sensitivity of the method was close to that of the detection of 10 PFU CBV particles. CBV RNA were extracted by NaI guandinium thiocyanate chioform method, and Compared with proteinase K phenol chloform and guanidium thiocyanate phenol extraction, the NaI extraction method was rapid, cheap and safe and this amplification effect was similar to the three methods. Viral RNA could be detected by RT PCR in the blood of CBV 5 of infected mouse on the 30 day of postinfection, and the viral RNA could be detected up to on the 30 day of postinfection. These results indicated that RT PCR technique was suitable for early diagnosis of CBV infection.