Research And Application Of Next-Generation Sequencing Technology Based On Multiple PCR In High-Throughput Detection Of Various Pathogens

Next-generation sequencing,also known as high-throughput detection technology,has a wide range of applications in clinical detection.This technology allows multispecies high-throughput sequencing at the same time.These sequences can come from microbial isolates of different patients or multiple infections from the same patient sample.In addition,this technique is generally considered as the most efficient method which was used for comprehensive evaluation of bacterial arrays.However,for very low abundance taxonomies in mixed species,only sequencing analysis is deficient in read depth,so that PCR amplification is required prior to Illumina sequencing.Therefore,based on the statistical analysis of CHINET(2017),this study selected 22 pathogens as the research goals which frequently appeared in mixed infection clinical samples and include 19 bacterium and 3 fungi.In order to accurately detect the above pathogens in clinical specimens,we developed a high-throughput detection platform based on multiplex PCR and next-generation sequencing.We designed 44 primers pairs for 22 clinical pathogens,and this primer mix was called"Clinical Pathogens Panel"(CPP).CPP was designed for sequencing on the Illumina Hi Seq 2500 platform,and 22 targeted pathogens can produce specific reads.Firstly,the nucleic acids of 22 clinical pathogens were used as templates,and 44 primers pairs were amplified by using PCR.The results showed that all primers had good specificity and sensitivity.The homogeneity of CPP was evaluated by multiplex PCR amplification and next-generation sequencing.The results showed that the uniformity values before adjustment of the primer concentration between 0 and 8.70,and the adjusted uniformity values between0.33 and 2.37.The adjusted CPP reads had a significant difference(P<0.001),which was proved that the primer concentration adjustment had a significant effect.Next,using the concentration-adjusted CPP as the primer,the multiplex PCR amplification system was respectively amplified with 19,22,25,and 28 cycles.The results showed that the primer dimer increased with increase of cycle number.But there were no dimeric bands on agarose gel electrophoresis,which can exclude the effect of primer dimer on CPP.Therefore,multiplex PCR cycles are available between 19 and 28.Then 12 kinds of broken methods were adopted to physically crush typical Gram-positive bacteria(S.aureus?E.faecium),Gram-negative bacteria(K.pneumoniae?E.coli)and fungi(C.albicans).The results showed that when the pathogen concentration was below 1 OD,0.5g(0.1mm:0.5mm=1:1)glass beads were used to break pathogens,and the cell wall breaking rates were higher than 95%.Real-time PCR(q PCR)technology was used to compare the nucleic acid extraction effects of three different commercially available kits.The results showed that the best choice was to use Mag Pure Circulating DNA Kit B with the nucleic acid automated extraction instrument.And on the basis of physical fragmentation,only the low concentration of S.aureus needs to be enzymatically hydrolyzed.At this time,the extracted nucleic acid is significantly different from the nucleic acid extracted without enzyme(P<0.05).In addition,based on the above physical fragmentation conditions and nucleic acid automated extraction platform,10~6,10~5,10~4,10~3,10~2 cfu/m L of S.aureus and C.albicans were respectively subjected to nucleic acid extraction.It showed that when the concentration of pathogen was 10~6 cfu/m L,the nucleic acid extraction efficiency of S.aureus and C.albicans respectively reached 85%and 90%.And when the concentration of pathogen was between10~6?10~2 cfu/m L,the nucleic acid extraction efficiency of C.albicans and S.aureus decreased with decreasing concentration.However,at 10~4 cfu/m L,the nucleic acid extraction efficiency of S.aureus was at least 11.3%.Finally,the next-generation sequencing based on multiplex PCR was performed on 11sputum samples.The positive rate of clinical culture was 72.73%(8/11),and the positive rate of t m PCR-NGS results was 100%.Only one of them was a single infection,and the others were mixed infections.And the highest occurrence pathogen was S.maltophilia(7/11).11 blood samples were tested by next-generation sequencing based on multiplex PCR.The results showed that the sensitivity of this method was higher than q PCR,Sanger sequencing and clinical culture.All in all,our work showed that NGS based on multiplex PCR can detect 22 pathogens in clinical samples.The NGS results of CPP have a certain correlation with the initial concentration of amplicon.And this method has the following advantages:first,it can be used for qualitative and quantitative detection of clinical pathogens;in addition,it can identify dominant and non-dominant species in the same sample;finally,the use of CPP for clinical diagnosis can reduce analysis time and cost,because multiple samples and different pathogen genes can be tested simultaneously.