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目的研究2种前处理方法对粘液型标本HPV DNA杂交结果的影响,为进一步提高临床检验质量和改进检测技术提供理论依据。方法收集100例妇科门诊患者粘液型宫颈脱落细胞标本,分别采用常规方法(生理盐水震荡法)和蛋白酶K消化法对脓性粘液型标本进行前处理,应用核酸分子快速导流杂交技术进行21种HPV亚型感染检测。研究不同前处理方法对HPV DNA浓度、纯度和分型结果的影响。结果①常规方法处理后所抽提的DNA纯度、含量分别为1.63±0.71和(96.35±13.15)μg/ml,蛋白酶K消化法处理后所抽提的DNA纯度、含量分别为1.80±0.52和(105.14±18.65)μg/ml,蛋白酶K消化法处理后检测的DNA纯度和含量分别高于常规法(P<0.01)。②常规法检测出现PCR反应抑制物的例数为4例,而蛋白酶K消化法则为0例。③常规法和蛋白酶K消化法HPV分型的总阳性率、单一感染率、多重感染率分别为28.0%、23.0%、5.0%和32.0%、26.0%、6.0%,两法一致性检验Kappa=0.888,结果具有较好的一致性。结论基于蛋白酶K消化法的HPV检测,对粘液型标本可获得质量较好的HPV DNA,无核酸丢失和污染,分型结果满意,可以有效去除PCR反应抑制物,操作简便,适合临床检验应用。
Objective To study the effect of two pretreatment methods on the results of HPV DNA hybridization in mucoid specimens and provide theoretical basis for further improving the quality of clinical tests and improving the detection techniques. Methods 100 specimens of mucous-type cervical exfoliated cells were collected from gynecological clinic patients. Purulent mucinous specimens were pretreated by routine method (saline concussion method) and digestion by proteinase K. The rapid flow-through hybridization technique was used for 21 Detection of HPV subtypes. The effect of different pretreatment methods on HPV DNA concentration, purity and typing results was studied. Results ① The purity of DNA extracted by conventional method was 1.63 ± 0.71 and (96.35 ± 13.15) μg / ml, respectively. The purity of DNA extracted by digestion with proteinase K was 1.80 ± 0.52 and ( 105.14 ± 18.65) μg / ml. The purity and content of DNA detected by digestion of proteinase K were higher than those of the conventional method (P <0.01). ② There were 4 cases of PCR reaction inhibitors detected by routine method, 0 cases of protease K digestion method. ③ The total positive rate, single infection rate and multiple infection rate of HPV typing by routine method and proteinase K digestion method were 28.0%, 23.0%, 5.0% and 32.0%, 26.0% and 6.0%, respectively. The consistency of two methods was Kappa = 0.888, the result has a good consistency. Conclusion Based on the HPV test of proteinase K digestion method, HPV DNA with good quality can be obtained from mucus samples without nucleic acid loss and contamination. The result of typing is satisfactory and the PCR reaction inhibitors can be effectively removed. The method is simple and easy to operate and is suitable for clinical application.