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Comparison of Two PCR-Based Human Papillomavirus Genotyping Methods

机译:两种基于PCR的人乳头瘤病毒基因分型方法的比较

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摘要

We compared two consensus primer PCR human papillomavirus (HPV) genotyping methods for the detection of individual HPV genotypes and carcinogenic HPV genotypes as a group, using a stratified sample of enrollment cervical specimens from sexually active women participating in the NCI/Costa Rica HPV16/18 Vaccine Efficacy Trial. For the SPF10 method, DNA was extracted from 0.1% of the cervical specimen by using a MagNA Pure LC instrument, a 65-bp region of the HPV L1 gene was targeted for PCR amplification by using SPF10 primers, and 25 genotypes were detected by reverse-line blot hybridization of the amplicons. For the Linear Array (LA) method, DNA was extracted from 0.5% of the cervical specimen by using an MDx robot, a 450-bp region of the HPV L1 gene was targeted for PCR amplification by using PGMY09/11 L1 primers, and 37 genotypes were detected by reverse-line blot hybridization of the amplicons. Specimens (n = 1,427) for testing by the LA method were randomly selected from strata defined on the basis of enrollment test results from the SPF10 method, cytology, and Hybrid Capture 2. LA results were extrapolated to the trial cohort (n = 5,659). The LA and SPF10 methods detected 21 genotypes in common; HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73 were considered the carcinogenic HPV genotypes. There was no difference in the overall results for grouped detection of carcinogenic HPV by the SPF10 and LA methods (35.3% versus 35.9%, respectively; P = 0.5), with a 91.8% overall agreement and a kappa value of 0.82. In comparisons of individual HPV genotypes, the LA method detected significantly more HPV16, HPV18, HPV39, HPV58, HPV59, HPV66, and HPV68/73 and less HPV31 and HPV52 than the SPF10 method; inclusion of genotype-specific testing for HPV16 and HPV18 for those specimens testing positive for HPV by the SPF10 method but for which no individual HPV genotype was detected abrogated any differences between the LA and SPF10 methods. The LA method detected more carcinogenic-HPV-genotype infections per specimen than the SPF10 method (P < 0.001). In conclusion, the LA method and the SPF10 method with HPV16 and HPV18 genotype-specific detection among ungenotyped HPV-positive specimens were comparable for detection of HPV16 and HPV18, the two HPV genotypes targeted by current prophylactic HPV vaccines. Both approaches are suitable for monitoring the impact of HPV16/18 vaccines in clinical trials.
机译:我们比较了两种共有引物PCR人类乳头瘤病毒(HPV)基因型检测个体HPV基因型和致癌性HPV基因型的方法,该方法使用了分层样本的参与NCI /哥斯达黎加HPV16 / 18性活跃妇女的子宫颈样本疫苗效力试验。对于SPF10方法,使用MagNA Pure LC仪器从0.1%的宫颈标本中提取DNA,使用SPF10引物将HPV L1基因的65 bp区域用于PCR扩增,并通过反向检测25种基因型扩增子的在线印迹杂交。对于线性阵列(LA)方法,使用MDx机器人从0.5%的宫颈标本中提取DNA,使用PGMY09 / 11 L1引物将HPV L1基因的450 bp区域作为PCR扩增的靶标,并使用37通过扩增子的反向印迹杂交检测基因型。从根据SPF10方法,细胞学和Hybrid Capture 2的入选测试结果定义的分层中随机选择用于LA方法测试的标本(n = 1,427)。将LA结果外推至试验队列(n = 5,659) 。 LA和SPF10方法检测到21个共同的基因型。 HPV16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-66,-68和-73被认为是致癌的HPV基因型。 。通过SPF10和LA方法分组检测致癌性HPV的总体结果没有差异(分别为35.3%和35.9%; P = 0.5),总体一致性为91.8%,kappa值为0.82。与单个HPV基因型的比较中,与SPF10方法相比,LA方法检测出的HPV16,HPV18,HPV39,HPV58,HPV59,HPV66和HPV68 / 73明显多得多,而HPV31和HPV52较少;对于那些通过SPF10方法检测为HPV阳性但未检测到单个HPV基因型的标本,HPV16和HPV18的基因型特异性检测消除了LA和SPF10方法之间的任何差异。与SPF10方法相比,LA方法每个标本检测出更多的致癌HPV基因型感染(P <0.001)。总之,在未定型的HPV阳性标本中,采用HPV16和HPV18基因型特异性检测的LA方法和SPF10方法可用于检测HPV16和HPV18,这是当前预防性HPV疫苗靶向的两种HPV基因型。两种方法均适用于在临床试验中监测HPV16 / 18疫苗的影响。

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