| Objective: At present,cervical cancer ranks fourth in all cancer deaths and second in female cancer [1-12].With the rising incidence of cervical cancer and the rising mortality rate,people are increasingly concerned about cervical cancer.Studies have shown that the occurrence of cervical cancer is inextricably linked to the repeated infection of human papillomavirus(HPV)[1-12].Therefore,early screening of HPV plays an indispensable role in the early detection,early diagnosis and early treatment of cervical cancer.HPV is a circular double-stranded DNA virus [13-30],which is easy to invade the squamous columnar epithelial tissues such as the cervix and skin mucosa,and is infected through sexual transmission,indirect contact,mother-to-child transmission,and iatrogenic infection [31-35]..Because different genotypes lead to different degrees of risk,there are three types of high,medium and low risk [57].Among them,low-risk type can lead to genital warts [60-68],high-risk type can cause cervical intraepithelial neoplasia [6,20,22,31,36-42],if not taken timely,there is a risk of progression to cervical cancer.[40,42-50].At present,there are many HPV screening technologies widely used at home and abroad.The most widely used molecular diagnostic technique is the membrane hybridization method,which can detect 21 genotypes,including HPV16,HPV18,HPV31,HPV33,HPV35,HPV39,HPV45,HPV51,HPV52,HPV56,HPV58,HPV68,medium-risk type HPV53,HPV66,low-risk type HPV6,HPV11,HPV42,HPV43,HPV44,HPV81.Qualitative analysis of HPV based on the strength of the detection site signal.The inherent defect is that HPV cannot be quantified,and it is impossible to judge the change of the disease during the later clinical treatment.With the deepening of research,new HPV detection technology-multiple fluorescence PCR intubation technology has emerged,which can detect the same high-risk type and more intermediate-risk types,such as HPV26,HPV73,HPV82,and parts.Low-risk type,a total of 21 genotypes.And can carry out viral load quantification for HPV infected people.In this study,the sensitivity and specificity of multiplex fluorescent PCR intubation techniques were evaluated by comparison with the currently widely used membrane hybridization method.Explore the role of HPV screening and the role of guidance in clinical treatment in the quantification of viral load.Method: 1.Selected subjects: 2018.01-2018.10,100 women who were admitted to the gynaecology clinic of the First Affiliated Hospital of China Medical University and were willing to receive HPV screening tests.The 100 patients had sexual intercourse bleeding,irregular vaginal bleeding,vaginal discharge abnormalities,The lower abdomen fell,pain and other reasons for the main complaint.Inclusion and exclusion criteria: 1),previous sexual life history,and can accept cervical mucosal tissue biopsy.2),non-pregnancy and menstruation,the mother can perform this study after 8 weeks of delivery.3),no history of cervical resection.4),recently did not use vaginal contraceptives,vaginal lubricants,vaginal antifungal drugs.5),asexual behavior within 24 hours before the test.6),no tumors and other consumptive diseases(the body is unable to bear the special circumstances such as the inspection process).2.Detection of HPV genotypes:(1)Sampling: The exfoliated cells at the junction of the cervical squamous columnar epithelium were brushed with a cervical sampler,placed in a sterile tube,and sealed for examination.(2)The DNA of the cervical mucosal epithelial cells of the patient is extracted by a nucleic acid extractor.(3)21 kinds of targeted gene primers,probes and DNA to be tested were used for loading,and detected by real-time quantitative PCR(RTQ-PCR).(4)Adjust the baseline starting point,end point,and threshold according to the RTQ-PCR image,and refer to the HPV subtype probe fluorescent labeling table to obtain the selected genotype.3.Calculation of HPV viral load: Through TOP3(single-copy gene,linear relationship of cervical exfoliated cells)based on copy number,and then measuring the number of cells in the sample,and then through the clinical sample verification and optimization of the calculation model,finally can be drawn Viral load per unit cell(10000).Result: 1.Of the 100 patients,99 were effectively enrolled.The positive samples were detected by multiplex fluorescent PCR tube technique in 22 cases(22.22%),and the negative samples were 77 cases(77.78%).The positive cases of membrane hybridization were 13 cases(13.13%),and the negative samples were 86 cases(86.87%).There were 11 cases with inconsistent results detected by the two methods.2.The sequencing results were used to confirm the results of 11 cases inconsistent between the two methods,and the results were all positive.Among them,the multiplex PCR protocol has one genotype result inconsistent(consistency is 96.3%),the qualitative detection sensitivity is 100%,and the specificity is 100%.Membrane hybridization assay showed that 13 genotypes were inconsistent(consistent 51.8%),qualitative detection sensitivity was 59.09%,specificity was 100%.3.Using multiplex fluorescent PCR tube technique to detect 11 cases of missed detection by membrane hybridization,9 cases were clinically tracked,1 case was treated with colposcopy as cervical cancer in situ,and 2 cases were pathologically examined as high-grade cervix.Intraepithelial neoplasia.4.There was a statistically significant difference between the multiplex PCR coupled technique and the membrane hybridization method in detecting qualitative results and viral load(p=0.020).Membrane hybridization was not statistically different between the genotypes and the viral load(p>0.05).5.There was a statistically significant difference between the different pathological grades and their viral load(p < 0.001).Conclusion: 1.The sensitivity and specificity of multiplex PCR coupled tube technology are significantly better than the currently widely used membrane hybridization method.2.Multiplex PCR PCR technology can quantitatively detect viral load and provide a basis for guiding clinical treatment. |
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