Evaluation And Application Of 16S High-throughput Sequencing For Detection Of Rodent-borne Pathogens

BackgroundRodent-borne disease is a kind of zoonosis that does great harm to humans.In China,rodent borne diseases mainly include hemorrhagic fever with renal syndrome,leptospirosis,plague,etc.With the increasingly close economic and cultural exchanges in the world,there have been the occurrence of a variety of new mouse borne diseases and the resurgence of old infectious diseases,such as Leptospira disease caused by Leptospira interrogans,Bartonella disease caused by Bartonella spp.,and tsutsugamushi disease caused by Orientia tsutsugamushi.These diseases spread rapidly,endanger people’s health,and seriously affect public health and safety.Quantitative polymerase chain reaction(qPCR)is the main method for monitoring mouse borne pathogens in China,which can detect one or several pathogenic bacteria.The isolation,culture and identification of bacteria are the "gold standard" for the diagnosis of bacterial infectious diseases.Anticoagulant and liver and spleen tissues are often used for in vitro culture.16S rRNA(16S ribosomal RNA)gene high-throughput sequencing can detect the bacterial community in rodents’ internal and external environments.Currently,the second-generation sequencing platform is the most commonly used.The second-generation Illumina sequencing platform is a sensitive and high-throughput pathogen detection method.The amount of sequencing data can accurately evaluate the relative abundance of bacteria,and can provide high-resolution detection with reference to the comprehensive database.The third-generation sequencing technology represented by nanopore sequencing has the characteristics of long detection fragment length and short detection time,which can directly and real-time detect and identify pathogenic bacteria in rodent viscera.Timely and accurate detection of pathogens is the key to the prevention and control of mouse borne diseases.Therefore,it is of great significance to establish an efficient,rapid,simple method that can simultaneously detect a variety of mouse borne diseases.In this study,fluorescence quantitative PCR and bacterial isolation and culture detection of murine pathogenic bacteria were selected as controls to evaluate the sensitivity,repeatability and accuracy of 16S high-throughput sequencing detection of murine pathogenic bacteria,which has application value for on-site detection and pathogen monitoring.ObjectivesIn this study,the sensitivity,repeatability and accuracy of 16S high-throughput sequencing method for the detection of mouse borne pathogens were evaluated by using fluorescence quantitative PCR and bacterial isolation and culture method as the control.The feasible detection methods for the implementation of comprehensive monitoring of mouse borne diseases were explored,and the references for the field detection of mouse borne pathogens were provided.Methods1.Laboratory simulation sample testing and evaluationThe standard strain of Bartonella grahamii(BG),a common mouse borne pathogen,was selected as the simulated sample,and the bacterial solution with different dilutions was isolated and cultured,and the number of BG colonies was counted;BG whole genome nucleic acid was extracted,and the copy number of BG nucleic acid at different dilutions was quantified by fluorescence quantitative PCR(qPCR);The BG base sequences(reads)with different dilutions were detected by 16S high-throughput sequencing,and the results of qPCR,16S high-throughput sequencing and isolation culture were compared.2.Field sample testing and method evaluationThe spleen and kidney tissue samples were collected aseptically from the rodents captured by night trapping method,and the whole genome was extracted.Six common rodent-borne pathogens including Bartonella spp.,Leptospira interrogans,Orientia tsutsugamushi,Rickettsia mooseri,Anaplasma phagocytophilum,and Francisella tularensis were detected by fluorescence quantitative polymerase chain reaction(qPCR)with TaqMan probe.Illumina sequencing and Nanopore sequencing after routine PCR amplification with universal primers for the 16S ribosomal RNA(16S rRNA)gene were employed to further detect the pathogens.Meanwhile,the spleen tissue was used for the isolation and culture of Bartonella in vitro.The results of qPCR,16S amplicon sequencing,and bacterial isolation and culture were compared.μLResults1.Use a turbidity meter to adjust the initial concentration of Bg suspension to 2.1 Mc Farland(MCF),dilute 10 times the series,and inoculate for cultivation.When diluted to 2.1× At 10-5 o’clock,there was 10 CFU on the plate,approaching the detection limit for bacterial isolation and cultivation.Extract Bg nucleic acids with different dilutions,draw standard curves with known concentrations of Bartonella plasmid standards to calculate the amount of Bg samples.When diluted to 10-5,the cycle threshold(Ct)value is 36.76,and the copy number concentration is 79.43 copies/μL.Perform Illumina sequencing and Nanopore sequencing on Bg nucleic acid.At a certain sequencing depth,the higher the template concentration,the more base sequences(reads)for classification analysis,indicating a certain trend.Reads used for classification in the diluted sample are significantly reduced.When diluted to 10-6,reads are as low as single digits or even undetectable.2.A total of 66 rodents of 8 species were captured,of which 31(47.0%)rodents were Apodemus uralensis,and the rest were Rattus norvegicus,Mus musculus,etc.The infection rates of common rodent-borne pathogens detected by qPCR were as follows:Bartonella 31.8%(21/66),Leptospira interrogans 1.5%(1/66),Orientia tsutsugamushi 1.5%(2/66),Rickettsia mooseri 3.0%(1/66),and Francisella tularensis 13.6%(9/66).A.phagocytophilum was not detected.The Illumina sequencing of 16S rRNA amplicon detected pathogens(mainly Bartonella)in the 23 samples passing the quality control.The Nanopore sequencing of 16S rRNA amplicon detected Bartonella in 11 samples passing the quality control,and did not detect the other 5 common rodent-borne pathogens.Bartonella was isolated from 11 spleen tissue samples,with the infection rate of 16.7%(11/66).Conclusions1.Monitoring and method evaluation of laboratory simulated samples and on-site samples have shown that isolation and cultivation have shortcomings such as difficulty in cultivation and low sensitivity.QPCR has good sensitivity and specificity,simple operation,but has the limitation of low throughput.16S high-throughput sequencing is more sensitive to the detection of low copy number pathogens,with the characteristics of high-throughput and no target,but there are microbial contamination and base mismatch errors.The three detection methods can complement each other and more comprehensively detect and identify mouse borne pathogens.2.Rodents in the Altay prefecture of Xinjiang can carry a variety of rodent-borne pathogens such as Bartonella.We should pay attention to and strengthen the prevention and control of related infectious diseases in this region.