聚合酶链反应标记输血传播病毒(TTV)地高辛素探针的初步应用

Detection of transfusion transmitted virus(TTV) DNA using DIG labeled probe prepared by polymerase chain reaction

用聚合酶链反应法（ＰＣＲ）制备地高辛素标记的输血传播病毒（ＴＴＶ）探针，并与巢式ＰＣＲ法（ｎＰＣＲ）比较。结果该探针具有ＴＴＶ特异性，其灵敏度为１０ｐｇＤＮＡ。应用该法检测１０８份非甲～庚型肝炎病人的血清标本，ＴＴＶＤＮＡ阳性率为１８５％（２０／１０８）；检测２２份病人的粪便标本，ＴＴＶＤＮＡ阳性率为２７３％（６／２２）。该法与ｎＰＣＲ法的总符合率为９６９％（１２６／１３０）。结论：用ＰＣＲ法直接制备地高辛素标记ＤＮＡ探针简便、快速、灵敏度高，特异性好。应用该法从６名病人的粪便中检出ＴＴＶＤＮＡ，提示ＴＴＶ有可能通过粪口途径传播。

Objective To study the sensitivity and the specificity of digoxigenin(DIG) labeled DNA probe for detection of TTV DNA.Methods\ The probe was directly prepared from products of polymerase chain reaction(PCR).TTV DNA was detected by dot blot hybridization,and then the sensitivity and the specificity of the probe was compared with that of the nested PCR(nPCR).Results\ The sensitivity of the DIG DNA probe was 10 pg TTV DNA.108 sera and 22 feces of patients with non A～G hepatitis were tested by dot blot hybridization.The positive rate of TTV DNA was 18 5%(20/108) for sera,and 27.3%(6/22) for feces,respectively.The total coincidence rate between the dot blot hybridization and the nPCR was 96.9%(126/130).Conclusion\ The direct prearation of DIG labeled TTV DNA probe by PCR is much faster and more simple than the traditional method of labeling.The presence of TTV in feces of patients with TTV infection indicates the possibility of fecal oral transmission of the virus.