Establishment And Application Of A High-throughput Detection Method For Common Respiratory Pathogens Based On Mass Spectrometry

Respiratory tract infections continue to present an enormous public health burden and pose a major threat to young children.A variety of pathogens can lead to respiratory tract infections and among the causative organisms,viruses account for more than 80%cases and bacteria are the leading cause of serious disease and deaths.Accurately grasping the pathogenic spectrum of respiratory tract infections can provide the basis for formulating the strategies of prevention and control for related infectious diseases.Clinically identifying pathogen in the early stage of disease,therefore selecting appropriate antibiotics accordingly is critical to control infections.Routinely used microbiological detection methods include culture,serological testing and immunoassay.However,such methods have limitations such as long turn-around time and low sensitivity.PCR based molecular methods have high sensitivity and less turn-around time but most of them cannot take into account the two key factor of pathogen coverage and sample throughput.Moreover,some excellent detection technologies are relatively expensive,which prevents them being used widely.In view of this,this project aims to establish a highly efficient and cost effective approach for respiratory pathogen detection.MassARRAY System,a detection platform combing matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS)with endpoint PCR,was used to identify pathogens in this study.By reviewing researches and guidelines on epidemiology and detection methods of respiratory pathogens,we chose pathogens that are common in and related to respiratory infections to be included in the methods.For each target pathogen,a gene that is specific to the species and conserved within the species was selected as the target gene.According to different kinds of pathogens,we designed four strategies to collect sequences,which were then aligned and clusted.On the basis of above analyses,assays were designed witn the conserved regions.Finally,two sets of assyas were developed:respiratory viruses-mass spectrometry,RV-MS and respiratory bacteria-mass spectrometry,RB-MS.The RV-MS method is a 24-plex method which can be used to simultaneously screen for 21 common respiratory viruses.The RB-MS method is a 12-plex method that can simultaneously detect and identify 11 key bacterial pathogens related to pneumonia,meningitis and pertussis.Specificity of the method was validated by testing confirmed clinical samples and isolates.The two methods could accurately identify the expected pathogens without cross-reactivity with other pathogens.Plasmids containing the target genes of each pathogen were 10-fold diluted and used to evaluate the analytical sensitivity of the two methods.For each of the target pathogens,the analytical sensitivity of the two methods is approximately 10 to 100 DNA copies.Such high sensitivity enables the identification of low-abundance pathogens in mixed infection samples.To evaluate the usefulness of the RV-MS method,we used this method and consensus PCR/RT-PCR methods to screen 336 nasal and throat swabs collected from adults or children with suspected viral acute respiratory tract infections.Excluding four RNase-P-negative samples,the RV-MS and consensus PCR/RT-PCR methods detected respiratory viruses in 92.5%(307/332)and 89.5%(297/332)of the samples,respectively.The two methods yielded identical results for 306(92.2%)samples,including negative results for 25 samples(7.5%)and positive results for 281 samples(84.6%).The RB-MS method is a 12-plex method that can simultaneously detect and identify 11 key bacterial pathogens related to pneumonia and meningitis.To evaluate the clinical effectiveness of this method,204 nasopharyngeal swabs from hospitalized children with suspected pneumonia were tested using this method.In total,81.9%(167/204)of the samples were positive for at least one of the 11 target pathogens.Among the 167 bacteria-positive samples,the rate of multiple infections was 55.7%(93/167).We used real-time PCR and nested PCR to confirm positive results,with identical results obtained for 81.4%(136/167)of the samples.For positive results that failed to be verified,we revalidated them using 3D digital PCR,a detection platform with superior sensitivity.The results indicated that such differences might attribute to the higher sensitivity of the RB-MS method to PCR-related methods.The RV-MS method and the RB-MS method are highly efficient method and show good values of application.Firstly,they can simultaneously screen 32 pathogens in two reactions,which covers most of the common pathogens responsible for respiratory tract infections.The new methods’ capability of multiplex is four to five times higher than multiplex real-time PCR,which can decrease the sample usage dramatically.Secondly,testing can be performed in 3 84-well plates and one technologist is capable of analyzing 380 clinical specimens within one working day.Thirdly,the two methods can be selected according to the actual situation,and the detection targets within the method can be flexibly increased or decreased.Moreover,for each sample,the cost of detecting 32 pathogens simultaneously is about 50 RMB,which means the detection cost of individual pathogen is lower than 2 RMB.This price is highly competitive among the current detection technologies.Last but not least,the novel methods are developed based on MALDI-TOF MS,but not confined to MassARRAY platform.Therefore,an appropriate mass spectrometry platform can be selected in practice,thus expanding the application range.In summary,the two methods established in this study can be powerful tools used for pathogen screening at an early stage of respiratory infections and for large-scale epidemiological studies.It also sets an example for the development of syndrome-related pathogen screening technologies.