摘要
目的比较两种不同核酸提取方法在人乳头瘤病毒(HPV)亚型检测中的应用,为实验室选择相应的核酸提取方法来获得有效的HPV结果提供依据。方法收集25例浸润性宫颈癌福尔马林固定石蜡包埋(FFPE)组织标本,采用粗提法和纯化法两种核酸提取方法提取DNA标本,并通过检测DNA浓度、纯度及内参β-Globin来评估DNA质量。将提取的DNA经PCR扩增后,应用PCR-反向点杂交法检测HPV亚型,分析两种提取方法HPV检测结果的有效性及一致性。结果两种提取方法均能获得适量的DNA用于HPV检测,粗提法较纯化法可获得较高的DNA浓度(P<0.05),但存有一定有机物及蛋白质污染。纯化法较粗提法获得的DNA纯度更高(P<0.05)。将粗提法和纯化法提取的DNA用于检测HPV感染,总HPV阳性率分别为92%和96%。采用粗提法提取的DNA标本高危型HPV(HR-HPV)阳性率低于纯化法(92%vs.96%),但单一型HPV16阳性率高于纯化法(44%vs.36%);与纯化法相比,粗提法提取的DNA无法检测出更多型别的混合感染,而FFPE组织标本经纯化法分离后可检测出HPV33、HPV53、HPV58等多型别感染。结论采用粗提法和纯化法进行FFPE组织核酸提取,均可获得足量DNA用于HPV亚型检测,且HR-HPV阳性率达90%以上。粗提法具有操作简便、耗时较短、成本较低等优势,能够获得有效的HPV结果,但对于HPV多型别感染的检测表现欠佳。
Objective To compare the application of two different nucleic acid extraction methods in human papillomavirus(HPV)typing detection,to provide a basis for laboratories to select corresponding nucleic acid extraction methods to obtain effective HPV results.Methods Collected formalin-fixed paraffin-embedded(FFPE)tissue specimens from 25 cases of invasive cervical cancer.DNA samples were extracted by two nucleic acid extraction methods:crude extraction and purification.DNA quality was assessed by detecting DNA concentration,purity and internal referenceβ-Globin.After the extracted DNA was amplified by PCR,HPV typing was detected by PCR-reverse dot blot method.The validity and consistency of the HPV detection results of the two extraction methods were analyzed.Results The two nucleic acid extraction methods could obtain the right amount of DNA for HPV detection.Compared with the purification method,the crude extraction method could obtain higher DNA concentration(P<0.05),but there were certain organic and protein contamination.The purity of DNA obtained by the purification method was higher than that obtained by the crude extraction method(P<0.05).DNA extraction by crude extraction and purification were used to detected HPV infection,and the total HPV positive rates were 92%and 96%respectively.The positive rate of high-risk HPV(HR-HPV)in DNA samples extracted by crude extraction method was lower than that by purification method(92%vs.96%),but the positive rate of single type HPV16 was higher than purification method(44%vs.36%).Compared with the purification method,the DNA extracted by the crude extraction method could not detect more types of mixed infection,and the FFPE tissue specimens separated by the purification method could detect HPV33,HPV53,HPV58 and other multi-type infections.Conclusion Nucleic acid extraction from FFPE tissue by crude extraction and purification methods can obtain sufficient DNA for HPV typing detection.And HR-HPV positive rate more than 90%.The crude extraction method has the advantages o
作者
王诚
童玲玲
刘千琪
况薇
WANG Cheng;TONG Lingling;LIU Qianqi;KUANG Wei(Department of Pathology,West China Second Hospital,Sichuan University/Key Laboratory of Birth Defects and Related Diseases of Women and Children,Ministry of Education,Chengdu,Sichuan 610011,China)
出处
《检验医学与临床》
CAS
2022年第16期2203-2206,2210,共5页
Laboratory Medicine and Clinic
