Methods For Point-Of-Care Testing Of Pathogen Nucleic Acids Based On Invasive Reactions

Point-of-care testing(POCT is a technical means to detect samples at the sampling site by using portable detection devices and matching reagents,and nonprofessionals can carry out experimental operations and interpret the results.In recent years,nucleic acid detection technology has played an important role in the early diagnosis of pathogens and epidemic prevention and control.Due to short detection window period,high sensitivity and high specificity,nucleic acid detection technology has become a classic method for the screening and diagnosis of pathogens.Quantitative real-time PCR(qPCR,a traditional technique,is used for pathogens detection in most cases.However,this method requires a standardized laboratory with sample extraction,reagent preparation,and amplification detection areas,and requires professional technicians to process and analyze results.In addition,this method uses fluorescent probes or fluorescent dyes to detect amplicons,which relies on a complex and expensive fluorescence reading instrument.And the fluorescence reading instrument is bulky and not easy to carry,making this method difficult to be promoted and used in primary medical institutions.Thus,nucleic acid detection technology based on qPCR does not meet the requirements of POCT.To solve these problems,our study firstly established a vacuum self-injection microfluidic chip for multiple detection,which coupled reverse transcriptaserecombinase polymerase amplification(RT-RPA)with invasive reaction(Invader).Reaction reagents were integrated in the microfluidic chip,and then carried out the reaction in a portable fluorescence detection device.Finally,fluorescence results were read by a mobile phone.This method realizes multiple detection of 8 kinds of infectious pathogen nucleic acids with high sensitivity and specificity.The target RNA as low as 5 copies/reaction can be detected.And reaction chambers of different targets could not occur cross-reaction with good specificity.This method provides a POCT platform for "sample-in-result-out" to detect pathogen nucleic acids.Based on the previously established method of closed-tube visualization detection,our study makes PCR reaction and Invader reaction synchronous by optimizing the reaction conditions to improve the sensitivity of detection.In addition,our study also expands the application range of this method,establishing the visualized detection for 8 bacterial pathogens with a sensitivity of 10 copies/reaction.However,virus RNA could not be detected directly and should be reverse-transcribed into cDNA in advance,which caused tedious and time-consuming operations.Thus,by screening reverse transcriptases,investigating the compatibility of reverse transcriptases in the reaction systems and optimizing the reaction systems,a visualized new method for viruses RNA detection in a closed tube was established.SARS-CoV-2 and three variants RNA were detected,and N gene and ORFlab gene RNA can be detected respectively with a sensitivity of 5 copies/reaction.In addition,50 clinical samples were detected by using the proposed method and RT-PCR.The results of both methods have a 100%consistency,indicating that our proposed method has a good application prospect in clinical detection.In summary,our study has established methods based on invasive reactions for pathogen nucleic acids detection,which have the characteristics of simple operation,high sensitivity,high specificity,closed tube detection,convenient portability,etc.Our proposed methods meet the requirements of POCT and apply to different occasions including schools,grass-roots hospitals,and fever clinics.Our study provides new strategies for pathogen nucleic acids rapid screening,which have great clinical application value.