Development Of Multiple Quantitative Real-Time PCR Assay For Detection Of Respiratory Pathogens And Epidemiological Characteristics Of Human Adenovirus 2,3 And 7 Genetypes

Part One Establishment and evaluation of a quadruple quantitative real-time PCR assay for simultaneous detection of human coronavirus subtypesObjective:The early clinical symptoms of severe acute respiratory syndrome coronavirus 2（SARS-Co V-2）and seasonal HCo V infections are similar,so rapid and accurate identifcation of the subtypes of HCo Vs is crucial for early diagnosis,early treatment,prevention and control of these infections.However,current multiplex molecular diagnostic techniques for HCo V subtypes including SARS-Co V-2 are limited.So,the objective of the study is to develop a quadruple quantitative real-time PCR assay（qq-PCR）for simultaneous detection of SARS-Co V-2,SARS-Co V and the four seasonal HCo Vs（HCo V-229E,HCo V-NL63,HCo V-HKU1 and HCo V-OC43）.Methods:We designed primers and probes specifc for the S and N genes of SARS-Co V-2,the N gene of severe acute respiratory syndrome coronavirus（SARS-Co V）,and the ORF1ab gene of four seasonal HCo Vs,as well as the human B2M gene product.We developed and optimized a quadruple quantitative real-time PCR assay（qq-PCR）for simultaneous detection of SARS-Co V-2,SARS-Co V and four seasonal HCo Vs.This assay was further tested for specifcity and sensitivity,and validated using 184 clinical samples.Results:The limit of detection of the qq-PCR assay was in the range2.5×10¹to 6.5×10¹ copies/μL for each gene and no cross-reactivity with other common respiratory viruses was observed.The intra-assay and inter-assay coefficients of variation were 0.5-2%.The qq-PCR assay had a 91.9%sensitivity and 100.0%specifcity for SARS-Co V-2 and a 95.7%sensitivity and 100%specifcity for seasonal HCo Vs,using the approved commercial kits as the reference.Compared to the commercial kits,total detection consistency was 98.4%（181/184）for SARS-Co V-2 and 98.6%（142/144）for seasonal HCo Vs.Conclusions:With the advantages of sensitivity,specifcity,rapid detection,cost-efectiveness,and convenience,this qq-PCR assay has potential for clinical use for rapid discrimination between SARS-Co V-2 and seasonal HCo Vs.Part Two Simultaneous detection of 9 respiratory pathogens using a newly developed multiple quantitative real-time PCR panel based on an automatic molecular detection and analysis systemObjective:This study aims to establish a new multiplex real-time PCR（m RT-PCR）panel based on an automatic molecular detection and analysis system（Auto Molec system）for the combined detection of 9 respiratory pathogens and to provide a high accuracy,simple operation,rapid processing,continuous sample introduction and on-call testing method for diagnosis of respiratory infections.Methods:The m RT-PCR panel consisted of three separate internally controlled RT-PCR assays targeting 9 respiratory pathogens.The contents were as follows:assay 1:MP,CP,ADV and human globin gene（internal control）;assay 2:HMPV,Flu B,RSV and human 18S RNA（internal control）;assay 3:HPIV 1,HPIV 2,HPIV 3 and human 18S RNA（internal control）.Specific primers and probes were designed from the conserved regions of 9respiratory pathogens,human globin gene and human 18S RNA using oligo7.6.The m RT-PCR panel was developed and optimized,then combined with Auto Molec system.This system was further tested for specifcity,sensitivity and reproducibility,and it’s clinical performance was evaluated by comparison with an approved commercial kit,using 517 clinical samples.Results:The limit of detection of the Auto Molec m RT-PCR panel ranged from 4×10^-4～3.3 TCID50/m L and no cross-reaction with common respiratory pathogens was observed.The CVs of Ct value for reproducibility of low positive and moderately positive plasmids were both≤5%and results produced for the negative control were all negative.The Auto Molec m RT-PCR panel had 99.09%sensitivity and 100.0%specificity and overall detection consistency was 99.61%,making it comparable to that of the commercial kit.Conclusions:We successfully established an m RT-PCR panel based on the Auto Molec system for the combined detection of 9 respiratory pathogens.The novel system has the advantages of high accuracy,simple operation,rapid processing,continuous sample introduction and on-call testing and may have great potential for routine screening of respiratory infections in China.Part Three Epidemiological characteristics of human adenovirus 2,3 and 7 genetypes in hospitalized children with respiratory infection in a hospital of pediatric in Hebei Province from 2018 to 2020Objective:To investigate the epidemiological characteristics of human adenovirus（ADV）2,3 and 7 in hospitalized children with respiratory infection.Methods:A total of 25,686 children with respiratory infection hospitalized at Children’s Hospital of Hebei Province from January 2018 to December 2020 were retrospectively included.Deep sputum or nasopharyngeal aspirates of those children were collected.Then thirteen common respiratory pathogens were detected by multiplex PCR.510 ADV positive specimens were randomly selected via random number and classified for type 2,3 and 7 using a multiplex real-time quantitative PCR.SPSS 21.0software was used to perform all of the statistical analyses.Enumeration data were expressed by frequency and percentage.χ² test was used for comparison between groups.Results:The ADV-positive rate was 7.99%（2052/25686）.Children at age 3～6 years had the highest ADV-positive rate（11.44%）.The ADV-positive rate in 2019 was highest（10.64%）.Among the 510 ADV-positive specimens,the proportion of type 3 was the highest（31.16%）,followed by type 7（21.37%）and type 2（11.18%）.The rate of type 2 in 2019 was significantly lower than that in 2018 and 2020（χ²=8.954 and 16.354,P=0.003 and<0.001）,while the rate of type 3 was significantly higher than that in 2018 and 2020（χ²=5.248 and 4.811,P=0.022 and 0.028）.The rate of type 2,type 3 and type 7were lowest in winter,spring and autumn,respectively.The rate of type 2increased significantly in autumn and the rate of type 3 and type 7 increased significantly in winter.The co-detection rate of ADV with other respiratory pathogens was 43.33%（221/510）.Among,the co-detection rate of type 3 was highest（47.32%）,and the co-detection rate of type 2,3 and 7 was significantly higher than the alone detection rate（χ²=20.438,P<0.001;χ²=42.105,P<0.001;χ²=27.573,P<0.001）.The proportion of severe pneumonia in children with type 7 positive（15.89%）was higher than that in children with non-type 7positive（8.23%）（χ²=5.260,P=0.022）.Conclusions:ADV is one of the important pathogens of children with respiratory infection in Children’s Hospital of Hebei Province.The susceptible population of ADV is preschool children aged 3 to 6 years.ADV often co-detects with other respiratory pathogens.Type 3 and 7 is likely to be the dominant genotypes in this region,and type 7 may be one of the risk factors of severe pneumonia in children.Total conclusions:We successfully established a qq-PCR assay for rapid discrimination between SARS-Co V-2 and seasonal HCo Vs and an m RT-PCR panel based on the Auto Molec system for the combined detection of 9respiratory pathogens,which may have great potential for routine screening of respiratory infections in China.We also found that ADV 3 and 7 typeis likely to be the dominant genotypes in Children’s Hospital of Hebei Province,and type 7 may be one of the risk factors of severe pneumonia in children.