多重实时PCR检测三种呼吸道病毒

目的建立多重实时PCR检测体系可同时快速检测引起人呼吸道感染的甲型流感病毒（IAV）、呼吸道合胞病毒（RSV）和SARS冠状病毒（SARS-CoV）。方法通过对PCR反应条件的优化，建立了同时检测IAV、RSV和SARS-CoV的多重实时PCR方法。结果经熔解曲线分析，证实了该法可同时或分别检测3种重要呼吸道病毒。检测灵敏度分别为10^0.7 TCID50／ml、1 TCID50／ml和10^-0.5 TCID50／ml。采用相同的反应体系和条件，分别对其他7种呼吸道病毒（包括乙型流感病毒，副流感病毒2型，柯萨奇病毒B3和B5血清型及腺病毒2、3和7血清型）进行扩增，均未检测出扩增产物。此种方法可在4h内完成。结论建立的多重实时PCR检测方法适用于3种重要呼吸道病毒的快速检测。     

多重PCR检测儿童呼吸道感染常见病毒

目的 对深圳、粤东地区儿童呼吸道病毒感染进行监测、分析,探讨其流行规律.方法 2006年6月-2008年6月,于汕头大学附属医院、深圳市第四人民医院、揭阳市人民医院采集毛细支气管炎、支气管肺炎、喘息支气管炎等的患儿咽拭子或鼻咽分泌物标本686份,多重PCR进行9种呼吸道病毒检测.结果 在686例标本中病毒阳性362例(52.77%),其中呼吸道感染患儿中呼吸道合胞病毒(RSV)感染占首位,达到31.22%(113/362),其次是鼻病毒(RHV)为16.85%(61/362),最低是流感病毒B型(IVB)占0.83%(3/362),流感病毒A型(IVA)占14.36%(52/362),副流感病毒1型(PIV1)和副流感病毒3型(PIV3)分别占7.73%(28/362)和8.29%(30/362),腺病毒(AdV)达到9.67%(35/362),而人博卡病毒(hBOV)和人偏肺病毒(hMPV)分别占6.08%(22/362)和4.97%(18/362).结论 RSV、RHV及IVA是华南地区儿童呼吸道感染的主要病毒病原,混合感染以RSV、IVA联合其他病毒感染为主,诊治时应根据所感染病原体制定有针对性的措施.     

多重实时荧光定量PCR对9种常见呼吸道病原体检测

目的探讨多重实时荧光定量PCR(MRT-PCR)检测9种常见呼吸道病原体的临床应用价值。方法采用Primer Express3.0(ABI)和Primer3 Input 4.0设计引物和探针建立MRT-PCR法,通过构建质粒标准品分析该方法的最低检出限和特异性。并对204例临床标本中副流感病毒1、2、3型、肺炎支原体、肺炎衣原体、呼吸道合胞病毒、腺病毒、甲型流感病毒、乙型流感病毒进行回顾性检测。结果 MRT-PCR法检测病原体的最低检出限达103copies/m L,特异性达100%,无交叉反应。204例咽拭子标本中,MRT-PCR检出呼吸道合胞病毒23例,副流感病毒1、2、3型13例,腺病毒15例,肺炎支原体15例,肺炎衣原体3例,甲型流感病毒13例与乙型流感病毒6例。该结果与其他试剂报告结果完全一致。结论多重实时荧光定量PCR具有快速、准确、特异性强等特点,在呼吸道病原体检测方面有重要价值。     

2010—2012年韶关市儿童呼吸道感染病毒病原学分析

目的探讨韶关市儿童常见呼吸道感染的病原学特点和分布特征。方法采集2010年7月至2012年7月因呼吸道感染于粤北人民医院的住院患者呼吸道标本171份,采用荧光定量PCR方法,对呼吸道标本同时进行甲型流感病毒（FluA）,乙型流感病毒（FluB）,腺病毒（ADV）,博卡病毒（BoV）,副流感病毒1型（PIV1）、2型（PIV2）、3型（PIV3）,鼻病毒（HRV）,呼吸道合胞病毒（RSV）,冠状病毒229E、0C43、HKUl、NL63,偏肺病毒（MPV）等14种常见呼吸道病毒核酸检测。结果171份标本中检出阳性标本93份,核酸阳性率为54．4％（93／171）,其中FluA占首位,阳性率为8．2％（14／171）,其他依次为ADV7．6％（13／171）,HRV7．6％（13／171）,PIVI／II／III 7．0％（12／171）,RSV 6.4％（11／171）,FluB5．8％（10／171）,BoV5．3％（9／171）,MPV3．5％（6／171）,冠状病毒（HCoV—OC43／HKUl）2．9％（5／171）。不同性别问患儿呼吸道病毒阳性率差异无统计学意义（P〉0．05）。≥6月龄组阳性率最低（37．5％）,1—3岁年龄组阳性率最高（62．1％）。结论韶关地区儿童发热呼吸道病毒感染病例的病原体以甲型流感病毒、腺病毒和鼻病毒为主。     

呼吸道合胞病毒的分型研究

目的 了解北京地区１９９０￣１９９１年和１９９７￣１９９８年两个非连续的流行年中呼吸道合胞病毒（ＲＳＶ）Ａ、Ｂ亚型和基因型的流行情况。方法 用间接免疫荧光法（ＩＩＦ）检测ＲＳＶ阳性鼻咽分泌物（ＮＰＳ）标本或ＲＳＶ分离株,划分Ａ、Ｂ亚型。根据Ｎ基因片段的限制性酶切图型将ＲＳＶ分离株分成至少６个基因型ＮＰＩ－６ＮＰＩ,３和６属于Ｂ亚型,ＮＰ２４和５属于Ａ亚型。根据ＳＨ基因片段的核苷酸序列将Ａ亚型分离株     

阿瓦朗病毒和休斯病毒实时荧光RT-PCR检测方法研究

目的建立可以检测阿瓦朗病毒(Avalon virus,AVAV)和休斯病毒(Hughes virus,HUGV)两种内罗病毒的实时荧光定量RT-PCR检测方法,并进行初步的评价。方法收集、整理、比对、分析在公共数据库发布的两种病毒基因组核苷酸序列,确定检测靶标,设计特异性引物、探针,优化检测程序,建立实时荧光定量RT-PCR检测方法。利用体外转录技术制备的模拟样本、其他病毒感染标本、病毒株和正常人血标本比较评价所建方法的检测限、特异性、重复性特征。结果所建实时荧光定量RT-PCR检测方法可有效扩增检测AVAV和HUGA靶标RNA,检测限分别约为20拷贝/μl和70拷贝/μl,检测科萨努尔森林病毒、乙型流感病毒BV和BY型、甲型流感病毒H3N2、黄热病毒、乙型脑炎病毒、克里米亚-刚果出血热病毒、发热伴血小板减少综合征、内罗毕羊病毒和塔西那病毒样本无非特异性扩增,两种内罗病毒相互间无交叉反应,重复性比较分析显示变异系数小于2%。结论本研究建立的检测AVAV和HUGV的实时荧光定量RT-PCR方法,可用于临床样本检测和媒介生物、宿主动物标本筛查,便于病原的快速识别和疾病诊断。     

多重PCR技术检测小儿咽拭子呼吸道病毒的临床分析

目的 研究多重荧光聚合酶链式反应（PCR）技术对小儿咽拭子呼吸道病毒、病原菌检测的应用价值。方法 筛选2018年1月~12月到乌鲁木齐市社区服务门诊就诊的社区获得性肺炎（CAP）患儿524例,采用多重PCR技术检测患儿咽拭子病原菌,分析CAP病原菌的分布情况及其与年龄、季节等的关系。结果 CAP病原检测结果大部分属于病毒性,以乙型流感病毒、肺炎衣原体、肺炎支原体等为主,细菌类以肺炎克雷伯菌等为主;与患儿年龄、季节有关,3岁以下患儿仍是CAP的主要发病年龄段,尤其是婴幼儿最多,发病多集中在秋季和冬季;2种以上病原混合感染形式严峻。结论 多重PCR技术对小儿咽拭子病原菌检测的应用效果良好,快捷方便。     

脑心肌炎病毒 TaqMan real-time PCR检测方法的建立及应用

目的建立脑心肌炎病毒（ EMCV） TaqMan real-time PCR检测方法。方法根据GenBank中公布的EMCV 3D基因保守区段设计并合成1对引物和1条TaqMan 探针,建立EMCV TaqMan real-time PCR检测方法,且对体系进行优化;对该法进行灵敏性、特异性验证;采用建立的方法对98份猪血清样本进行检测,并与ELISA结果进行比较。结果建立的EMCV TaqMan real-time PCR检测方法线性关系较好,以质粒标准品构建的标准曲线相关系数R2为0．995;灵敏性比普通PCR高100倍,且仅能特异性检出 EMCV;对猪血清样本的检测与 ELISA 法检测结果符合率为98．0％。结论已建立了EMCV TaqMan real-time PCR检测方法,该法灵敏性高、特异性好,可用于EMCV的检测及定量分析。     

2021～2022年深圳市某医院常见呼吸道病毒流行病学特点

目的 分析2021年7月至2022年7月在我院就诊的急性呼吸道感染患者呼吸道病毒的感染情况,并比较多重荧光定量PCR法和胶体金法检测流感病毒的诊断效能。方法 收集我院1 263例急性呼吸道感染患者的鼻拭子标本,采用多重荧光定量PCR法进行检测,分析不同性别、年龄、季节6种常见呼吸道病毒的感染特点。随机抽取2022年1月至7月407例患者标本,采用胶体金法检测甲型流感病毒(influenza A virus, Flu A)和乙型流感病毒(influenza B virus, Flu B),并与多重荧光定量PCR法检测结果进行比较。结果 1 263例患者中,呼吸道病毒阳性检出率为21.54%(272/1 263),呼吸道合胞病毒(respiratory syncytial virus, RSV)阳性检出率最高,为8.55%(108/1 263)。不同性别患者阳性检出率差异无统计学意义(P>0.05)。秋季呼吸道病毒阳性检出率最高(P<0.001),其中RSV在秋季检出率最高,Flu A在夏季检出率最高、Flu B在春季检出率最高、副流感病毒Ⅰ型(parainfluenza vi...     

不同方法检测两种呼吸道病毒结果的比较

呼吸道合胞病毒 (ｒｅｓｐｉｒａｔｏｒｙｓｙｎｃｙｔｉａｌｖｉｒｕｓ,ＲＳＶ)、腺病毒(ａｄｅｎｏｖｉｒｕｓ ,ＡＤＶ)是引起儿科急性下呼吸道感染的两种重要病原。实验室提供及时和准确的病原检测报告 ,对临床明确病因、采取适当治疗手段和防止滥用抗生素有重要意义。自 90年代起 ,国外有报道将快速细胞培养和免疫组化的方法结合在一起检测病毒。本研究采用美国Ｃｈｅｍｉｃｏｎ公司生产并由ＷＨＯ认可的免疫荧光试剂 ,用直接涂片和快速细胞培养法分别检测呼吸道合胞病毒和腺病毒 ,并同时做常规的病毒分离 ,目的在于比较和评价不同方法…     

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.     

Detection of respiratory viruses in adults with respiratory tract infection using a multiplex PCR assay at a tertiary center

BackgroundRespiratory viruses (RVs) are among the most common pathogens for both upper and lower respiratory tract infections (RTIs). However, the viral epidemiology of RV-associated RTIs in adults has long been under-recognized. Through a sensitive molecular assay, it would be possible to have a better understanding of the epidemiology of RV-associated RTIs.Material and methodsRespiratory tract (RT) specimens from adults hospitalized due to RTIs were tested for RVs, using the multiplex PCR-based Luminex xTAG® Respiratory Viral Panel assay. A total of nineteen RVs, including influenza viruses and non-influenza respiratory viruses (NIRVs) were detected. Positive rates were compared using a chi-square test.ResultsA total of 2292 samples from adult patients hospitalized with RTIs were screened for RVs. The overall positive rate was 22%, with 17.8% samples positive for at least one NIRV. NIRVs had a higher positive rate in non-winter seasons. As many as 12.7% (46/363) of the samples collected through broncho-alveolar lavage and 20.5% (176/859) of the samples collected in ICUs were positive for RVs. Distribution of corona virus (CoV), human metapneumovirus (hMPV) and parainfluenza virus (PIV) demonstrated seasonal variation. Also, temperature was associated with the positive rates of specific viruses, including CoV, respiratory syncytial virus (RSV), hMPV and PIV.ConclusionRespiratory viruses, notably NIRVs, were frequently detected in adults hospitalized with RTIs. Several RVs were detected with distinctive seasonal variations. A substantial number of RVs were identified in lower RT specimens or from patients admitted to ICU, highlighting their important role in causing severe respiratory infection.     

Multiplex PCR system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis

ObjectivesTo provide a summary of evidence for the diagnostic accuracies of three multiplex PCR systems (mPCRs)—BioFire FilmArray RP (FilmArray), Nanosphere Verigene RV+ test (Verigene RV+) and Hologic Gen-Probe Prodesse assays—on the detection of viral respiratory infections.MethodsA comprehensive search up to 1 July 2017 was conducted on Medline and Embase for studies that utilized FilmArray, Verigene RV+ and Prodesse for diagnosis of viral respiratory infections. A summary of diagnostic accuracies for the following five viruses were calculated: influenza A virus (FluA), influenza B virus, respiratory syncytial virus, human metapneumovirus and adenovirus. Hierarchical summary receiver operating curves were used for estimating the viral detection performance per assay.ResultsTwenty studies of 5510 patient samples were eligible for analysis. Multiplex PCRs demonstrated high diagnostic accuracy, with area under the receiver operating characteristic curve (AUROC) equal to or more than 0.98 for all the above viruses except for adenovirus (AUROC 0.89). FilmArray, Verigene RV+ and ProFlu+ (the only Prodesse assay with enough data) demonstrated a summary sensitivity for FluA of 0.911 (95% confidence interval, 0.848–0.949), 0.949 (95% confidence interval, 0.882–0.979) and 0.954 (95% confidence interval, 0.871–0.985), respectively. The three mPCRs were comparable in terms of detection of FluA.ConclusionsPoint estimates calculated from eligible studies showed that the three mPCRs (FilmArray, Verigene RV+ and ProFlu+) are highly accurate and may provide important diagnostic information for early identification of respiratory virus infections. In patients with low pretest probability for FluA, these three mPCRs can predict a low possibility of infection and may justify withholding empirical antiviral treatments.     

A prospective study of prevalence of respiratory viruses causing acute respiratory infection in pediatric in-patients during pre- COVID times

PurposeTo find out the prevalence of respiratory viruses causing Acute Respiratory Infection in pediatric in-patients during Pre-COVID times.MethodsNasal swabs were collected from children in the age group of 1 month–16 years who were admitted at our hospital with Acute Respiratory Infection. Samples were subjected to nucleic acid extraction and Real time polymerase chain reaction to detect 16 RNA viruses and 2 DNA viruses. The results were interpreted in context of most prevalent viruses detected, their seasonal distribution, co-infecting viruses, co-morbidities in patients with effect thereof and use and effect of antibiotics in those positive for viral etiology.ResultsOf the 250 children recruited in the study, viral pathogen was detected in 74% cases. RSV was the most common virus detected with 36.2% positivity (92/254) followed by rhino/entero (19.2%, 49/254), PIV 1,2,3,4 (9.4%, 24/254), Influenza A,B,C (8.2%, 21/254), adenovirus & HBoV (6.2%, 16/254), coronavirus HKU1, NL63, OC43, 229E (4.3%, 11/254), H1N1 (4.7%, 12/254) and hMPV (0.7%, 2/254). Co-infection with 2 or more viruses was seen in 34% cases. Among the cases on whom antibiotics were started, they were withdrawn following test results in 42.3% of the cases.ConclusionThe prevalence of viral etiology is high amongst children especially ≤2 years. RSV, rhino/enterovirus, PIV 1,2,3,4 and Influenza virus were more prevalent than others. Rapid, early detection of virus with multiplex PCR will help in early cohorting of the patients thus reducing nosocomial spread of these viruses and prevent injudicious use of antibiotics.     

Species-specific identification of variola, monkeypox, cowpox, and vaccinia viruses by multiplex real-time PCR assay

A method of one-stage rapid identification of variola (VARV), monkeypox (MPXV), cowpox (CPXV), and vaccinia (VACV) viruses, pathogenic for humans, utilizing multiplex real-time TaqMan PCR (MuRT-PCR) assay was developed. Four pairs of oligonucleotide primers and four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were concurrently used for MuRT-PCR assay. The hybridization probe specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, TAMRA/BHQ2; CPXV-specific, JOE/BHQ1; VACV-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 29 strains belonging to six orthopoxvirus species as well as the DNA samples isolated from archive clinical specimens of human smallpox cases and experimental specimens isolated from CPXV-infected mice and MPXV-infected marmot.     

自动巢式多重PCR系统在呼吸道感染病例中快速检测呼吸道病原体的应用

目的 探讨自动巢式多重PCR系统在呼吸道感染病原体快速诊断中的应用价值.方法 2016年11月至2017年3月收集120例呼吸道感染患者的呼吸道标本和临床流行病学资料,利用自动巢式多重PCR系统对120份鼻咽拭子标本进行病原检测,对检测结果和临床资料进行统计学分析.结果 120份标本中单一病原检出68份,2种及2种以上病原混合检出22份,总检出率为75.0％(90/120),检出结果中以甲型流感病毒、呼吸道合胞病毒、百日咳杆菌为主.结论 自动巢式多重PCR系统敏感度高、特异性强,对呼吸道感染患者的病原学快速诊断具有重要价值.     

Respiratory etiological surveillance among quarantined patients with suspected lower respiratory tract infection at a medical center in southern Taiwan during COVID-19 pandemic

BackgroundA comprehensive study of respiratory pathogens was conducted in an area with a low prevalence of COVID-19 among the adults quarantined at a tertiary hospital.MethodsFrom March to May 2020, 201 patients suspected lower respiratory tract infection (LRTI) were surveyed for etiologies by multiplex polymerase chain reaction (PCR: FilmArray TM Respiratory Panel) test combination with cultural method, viral antigen detection and serologic surveys.ResultsTotal 201 patients tested with FilmArray TM Respiratory Panel were enrolled, of which 68.2% had sputum bacterial culture, 86.1% had pneumococcus and Legionella urine antigen test. Their median age was 72.0 year-old with multiple comorbidities, and 11.4% were nursing home residents. Bacteria accounted for 59.7% of identified pathogens. Atypical pathogens were identified in 31.3% of total pathogens, of which viruses accounted for 23.9%. In comparison to patients with bacterial infection, patients with atypical pathogens were younger (median= 77.2 vs 67.1, years, P = 0.017) and had shorter length of hospital (8.0 vs 4.5, days, P = 0.007).ConclusionsPatients with LRTI caused by atypical pathogens was indistinguishable from those with bacterial pathogens by clinical manifestations or biomarkers. Multiplex PCR providing rapid diagnosis of atypical pathogens enhance patient care and decision making when rate of sputum culture sampling was low in quarantine ward during pandemic.