标本保存液残留量对人乳头瘤病毒基因分型检测的影响

Effect of preserved solution on human papillomavirus DNA detection and genotyping

目的 观察人乳头瘤病毒（human papillomavirus,HPV）检测核酸提取过程中，弃上清液后标本保存液残留量对HPV基因分型检测结果的影响。 方法 采用流式荧光技术检测生殖道泌尿道分泌物HPV基因分型，根据弃上清液后标本保存液残留量分为4组：5 μL、20 μL、40 μL、80 μL组，进行扩增检测后再将上述5 μL组的10个样本的剩余DNA提取液再一分为二：一组加标本保存液80 μL（保存液组），一组加纯水80 μL（纯水组），进行扩增、杂交和检测，通过基因检测信号值和检出率以及电泳来评估标本保存液残留量对检测结果的影响。 结果 在HPV核酸提取过程中，弃上清液后HPV标本保存液残留量20 μL与5 μL组相比，内参和靶基因的检测信号值和检出率无差异（ P 〉0.05）；标本保存液残留量40 μL和80 μL组与5 μL组相比，靶基因的检测信号值和检出率均有显著差异（ P =0.0138, P =0.0001）；此外，纯水组，内参和靶基因的检测信号值均大于150，检出率100%，而加标本保存液组的检出率0%，两组的检测信号值和检出率有显著差异（靶基因 P = 0.0011 ,内参基因 P 〈0.0001）。 结论 在HPV核酸提取过程中，弃上清液后标本保存液残留量对HPV基因分型检测结果影响较大，标本保存液可抑制PCR的扩增而影响检测结果导致假阴性，残留量在20 μL以下才能保证检测结果的准确性。

Objective： To observe the effect of the residue of specimen preservation solution on the result of human papillomavirus （HPV） DNA detection and genotyping,in the process of nucleic acid extraction. Methods ： Flow cytometry was used to detect HPV genotypes of genitourinary tract secretions.According to the residual amount of preservative solution after discarding the supernatant,the samples were divided into 4 groups：5μL,20μL,40μL and 80μL.The remaining DNA extractions of the 5 μL group （10 samples） were further divided into two groups：80 μL stock solution added in preservation solution group,but 80 μL pure water added in pure water group.All groups were amplified,hybridized and detected.To assess the impact of sample preservation solution residue on the test results by the gene detection signal value,detection rate and electrophoresis. Results ： In the process of HPV nucleic acid extraction,there were no statistics difference （ P 〉 0.05 ） between the 5μL and 20μL group for the detection signal value and the detection rate of the internal reference and target genes.Compared with the 5μL group,the 40μL and 80μL group show lower detection signal value and detection rate of target genes（ P =0.0138, P =0.0001）.In addition,in the pure water group,the detection signal values of the internal reference and target genes of all samples exceed 150 and the detection rate was 100%;while the detection rate was 0% in preservation solution group,and there were significant difference in the detection signal value and the detection rate between pure water group and preservation solution group. Conclusion ： In the process of DNA nucleic acid extraction,the residual amount of the sample preservation solution after discarding the supernatant greatly affects the HPV genotyping test results,and the sample preservation solution can inhibit the PCR amplification resulting in false negatives.To ensure the accuracy of test results,the residual amount should be less than 20 μL.