Study On RPA Nucleic Acid Visual Point-of-care Test Technology

Recombinant enzyme polymerase amplification technology(RPA)is a new thermostatic in vitro nucleic acid amplification technology which USES recombinant enzyme and single chain binding protein to achieve the specific combination of primers and templates at room temperature in order to replace the denaturation and renaturation process in the traditional PCR thermal cycle.In this study,a detection method of transgenic maize TC1507 was established based on RPA technology,which can quickly detect the conservative section of transgenic maize TC1507 at a constant temperature of 36.7?,with good specificity,and the detection sensitivity is up to 10-2ng/ Libya,which is suitable for the rapid detection of transgenic maize TC1507 and its products in the grass-roots laboratory and on the spot;In addition,for Staphylococcus aureus(SAU),a rapid detection method based on recombinant enzyme polymerase mediated isothermal amplification technique(RPA)was established.Method according to the design of staphylococcus aureus gene conserved sequence,and the two pairs of primers specific oligonucleotide probe to positive staphylococcus aureus strains and other 7 kinds of the staphylococcus aureus genome DNA as a template,inspects the detection sensitivity and specificity of the method.The results RPA rapid detection of mycobacterium tuberculosis method only needs 5 min,the detection sensitivity of 10-2 cfu/m L;All 7 kinds of non-staphylococcus aureus could not be amplified,and their specificity was strong.This study established a rapid RPA detection method for staphylococcus aureus,which has the advantages of fast,simple and low cost,and provides a new tool for rapid detection of staphylococcus aureus;Recombinase Polymerase Amplification technology is a new thermostatic in vitro nucleic acid amplification technology that uses recombinase and single-stranded binding proteins at room temperature to achieve specific binding of primers and templates instead of the denaturation and replication process in the conventional PCR thermal cycle.In this study,a method based on RPA technology was developed for the detection of TC1507 in transgenic maize,which can rapidly detect the conserved region of TC1507 in transgenic maize at 36.7?,with good specificity and sensitivity of 10-2ng/?L.In addition,for Staphylococcus aureus,a method based on recombinase polymerase-mediated isothermal amplification(RPA)for rapid detection of S.aureus was established.The sensitivity and specificity of the method were investigated using two pairs of primers and a specific oligonucleotide probe designed according to the conserved sequence of the Staphylococcus aureus gene,and the genomic DNA of seven other non-Staphylococcus aureus strains.As a result,the RPA rapid detection method for Mycobacterium tuberculosis only takes 5 min,and the detection sensitivity is 101cfu/?L;the seven non-Staphylococcus aureus strains cannot be amplified,and the specificity is strong.In this study,a rapid RPA detection method for Staphylococcus aureus was established,which has the advantages of rapidity,simplicity and low cost,providing a new tool for the rapid detection of Staphylococcus aureus.Barley yellow mosaic virus(Ba YMV)is a widely distributed and highly destructive plant virus worldwide that can cause abrupt yield reductions and germplasm loss in barley.To solve this,a rapid and sensitive one-step reverse transcription recombinase polymerase amplification assay(RT-RPA)was developed to rapidly,accurately and effectively detect Ba YMV in barley.RPA-specific primers were designed based on the gene sequences of the conserved region of Ba YMV in the Gen Bank database;a total of eight viruses,Ba YMV,Ba MMV,BSMV,BYDV,BYSMV,BMMV,WSBMV,WSMV,and NCMV,were used for RT-RPA-specific validation;five template concentration gradients were designed to verify the sensitivity of the RT-RPA method,and the sensitivity of RT-RPA and RT-PCR were compared.The results showed that the RT-RPA assay was able to detect specific strips from Ba YMV samples;the specificity validation test showed did not detect strips except SMV.RT-RPA detected Ba YMV with a sensitivity of 10-3ng/?L and was more sensitive than RT-PCR.In summary,the RT-RPA method established in this study has high specificity and sensitivity for the detection of Ba YMV,and is suitable for rapid laboratory or field detection without special equipment.