Rapid Detection Of HPV Based On Paper-based Nucleic Acid Extraction

ObjectiveA rapid paper-based method for HPV DNA extraction was established based on the characteristics of easy access,low price,one-time use and portability of paper materials.Combined with recombinase polymerase amplification and electrochemiluminescence detection technology,the rapid,sensitive and specific detection of HPV DNA in clinical cervical exfoliated cell samples was realized.Methods1.A paper-based method for rapid extraction of HPV DNA from plasmids containing HPV type 16 gene fragment was established,and compared with the conventional boiling method.The extraction efficiency and amplification effect of HPV DNA from six different paper materials were compared,and the most suitable paper material for HPV DNA extraction and amplification was selected;the effects of different number of paper discs and extraction time on nucleic acid extraction and detection were studied;the sensitivity and repeatability of paper-based rapid extraction method of HPV DNA were discussed.2.The wax test strips with different nucleic acid binding area were designed to extract HPV-16 positive plasmid DNA.Combined with quantitative real-time PCR,the amplification effect of HPV DNA extracted by paper-based method and boiling method was compared,and the most suitable nucleic acid binding area was selected.The influence of the sample sizes on the nucleic acid amplification was investigated.The accuracy and applicability of paper-based method for nucleic acid extraction were analyzed by capturing of HPV DNA from cervical exfoliated cell samples with wax test strips.3.The method of Strip-RPA-ECL detection was studied with plasmid containing HPV 16 gene.RPA primers were designed to verify the feasibility of RPA with HPV DNA extracted from test strip.In order to explore the specificity and repeatability of the Strip-RPA-ECL method,and to analyze the accuracy and applicability in clinical application,the method was used to detect clinical HPV cervical exfoliated cell samples.Results1.A piece of qualitative filter paper disc with diameter of 6 mm can extract HPV 16 positive plasmid DNA under the conditions of binding time,washing time and elution time of 10 s.The results obtained by real-time fluorescent quantitative PCR are the same as those obtained by conventional boiling method.The detection limit of the paper-based rapid extraction method for HPV DNA was 8 × 102 copies/mL,the relative standard deviations of paper-based extraction method for 4×103 copies/mL and 5×105 copies/mL HPV 16 positive plasmids were 1.15%and 1.38%,which indicated that the method had good sensitivity and repeatability.2.HPV DNA was extracted from clinical cervical exfoliated cells with a piece of wax impregnated qualitative filter paper with a nucleic acid binding area of 2 mm×10 mm.The results of PCR amplification showed that 13 high risk HPV positive samples of 18 clinical samples were detected successfully.The results were consistent with the results of the conventional boiling method.It showed that the rapid extraction of HPV DNA paper base has clinical potential,and the extraction time of each sample is less than 1 min。3.When the molar ratio of esterified ruthenium to amino modified primer was 20:1,the labeling rate of Ru labeled primer was 87.21%.The peak area of electrochemiluminescence was 197432 after diluted 1000 times,which was enough to meet the needs of subsequent electrochemiluminescence detection.4.The detection limit of strip-RPA-ECL was 50 copies/ml,which was 16 times higher than qPCR.The relative standard deviations of 5×102 copies/ml and 5×105 copies/ml HPV16 positive plasmid ECL were 1.82%and 2.28%,the specificity,sensitivity and repeatability were good.The results showed that the electrochemi luminescence signal intensity of HPV 16 positive samples was significantly different from that of negative samples and other types of positive samples,which was consistent with the results of qPCR,indicating that strip-RPA-ECL method was suitable for HPV detection of clinical cervical exfoliated cells samples,and the time required for each sample from nucleic acid extraction to amplification was less than 1 h.ConclusionHPV DNA was extracted from cervical exfoliated cell samples by using strip,DNA template is directly eluted into the amplification reaction solution,which omits the steps of heating,centrifugation and pipetting in the traditional nucleic acid extraction process,simplifies the operation process and reduces cross contamination.RPA-ECL nucleic acid detection method has the advantages of rapid detection,simple operation,sensitivity and specificity.Therefore,the paper-based rapid extraction method of HPV DNA,combined with recombinant polymerase amplification and paper-based electrochemiluminescence detection technology,is expected to provide experimental basis for the development of point-of-care testing technology.