Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 10² copies/μL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.     

荧光RT-PCR对乙型流感病毒爆发的检测

目的探讨荧光RT-PCR方法对乙型流感病毒爆发的诊断价值。方法采集暴发性疫情的流感样病例的咽拭子,提取病毒RNA,PCR方法分别检测甲型和乙型流感病毒的核酸。然后,对核酸检测阳性标本进行MDCK培养,经红细胞凝集和凝集抑制试验对病毒进行鉴定和分型。结果 18份咽拭子中15份乙型流感病毒核酸呈阳性,甲型流感病毒未检出;15份乙型流感病毒核酸阳性标本经MDCK培养,9份标本发生细胞病变,经鉴定和分型,都属于乙型流感病毒Victoria系。结论 RT-PCR方法对乙型流感病毒爆发的检测具有快速、敏感和高度特异性的特点。本地区爆发的乙型流感病毒为乙型流感病毒Victoria系,提示在今后决定我国疫苗成份时应考虑地区流行的特点。     

Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/μL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.     

Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection

BackgroundReal-time PCR assay sensitivity is affected by the choice and concentrations of reaction mix constituents among other factors such as primers, probes, and analytical assay platforms. Commercially available reagent mixes facilitate PCR assay set-up with fewer steps and timeliness. However, determination of analytical assay framework is important for ready-to-use real-time PCR reagent systems for rapid, quantitative and accurate detection of bioterror pathogens such as Bacillus anthracis.MethodsIn this study, performance characteristics of five commercially available quantitative PCR reagent mixes were evaluated using TaqMan-based real-time PCR. The reagent systems were tested for compatibility on the ABI 7000 assay platform and compared for their distinctive analytical characteristics using the B. anthracis rpoB and pag gene real-time PCR assays.Results and conclusionsKnowledge of distinctive assay performance characteristics of commercially available qPCR reagent mixes is critical for carefully designing analytical assay systems. The ABI, ABgene and Eppendorf reagent systems performed consistently overall for the two TaqMan assays for B. anthracis detection that were used in the current study. However, the use of Eppendorf reagent system requires shorter thermal cycling time. In addition, while the ABI and Eppendorf systems have similar assay sensitivity for both the rpoB and pag assays, the Eppendorf system achieves the same with lower C_T values.     

登革病毒TaqManMGB实时荧光定量PCR检测方法的建立及初步应用

目的建立登革病毒TaqMan实时荧光定量PCR快速检测方法及应用于临床。方法根据1～4型登革病毒3’端非编码区的一段高度保守序列,设计一套型通用的引物和TaqManMGB探针,以4个血清型登革病毒标准株为标准,以日本乙脑病毒和丙肝病毒作阴性对照,以包含登革病毒2型标准株（DENV-2NGC株）3’非编码区349bD片段的质粒DNA作标准品,对引物和TaqManMGB探针的特异性、灵敏度进行分析,从而建立登革病毒实时荧光定量PCR检测方法。用该法对登革病毒野毒株和10份登革病毒患者血清进行检测。结果TaqMan实时荧光定量PCR检测的1～4型登革病毒标准株及野毒株均为阳性,日本乙脑病毒和丙肝病毒均为阴性;检测灵敏度可达到每反应2个基因拷贝;检测的10份登革患者血清样本中,8份检测结果为阳性。结论TaqManMGB实时荧光定量PCR方法是一个快速、特异性强、敏感性高的检测登革病毒的方法,适用于登革病毒的临床早期诊断。     

Development of a recombinase polymerase amplification fluorescence assay to detect feline coronavirus

Feline coronavirus (FCoV) is classified into two pathotypes: the avirulent feline enteric coronavirus (FECV), and the virulent feline infectious peritonitis virus (FIPV). Rapid pathogen detection, which is efficient and convenient, is the best approach for early confirmatory diagnosis. In this study, we first developed and evaluated a rapid recombinase polymerase amplification (RPA) detection method for FCoV that can detect FCoV within 15 min at 39 °C. The detection limit of that assay was 233 copies/μL DNA molecules per reaction. The specificity was high: it did not cross-react with canine distemper virus (CDV), canine coronavirus (CCoV), canine adenovirus (CAV), feline calicivirus (FCV), feline herpesvirus (FHV), or feline parvovirus (FPV). This assay was evaluated using 42 clinical samples (30 diarrhea samples and 12 ascites samples). The coincidence rate between FCoV-RPA and RT-qPCR for detection in clinical samples was 95.2%. In summary, FCoV-RPA analysis provides an efficient, rapid, and sensitive detection method for FCoV.     

Development and clinical evaluation of a novel fully automated qualitative PCR assay for the diagnosis of anogenital herpes simplex virus infection

Molecular detection of viral infections has the potential to improve microbial diagnostics, particularly with the emergence of rapid automated systems. We describe the design of the IDbox fully automated cassette-based system for nucleic acid extraction and real-time PCR amplification and perform a clinical evaluation for the diagnosis of genital herpes simplex infections. At optimal cutoff values determined by receiver-operator curves, the IDbox showed sensitivities of 94.9% (95% confidence interval [CI] 84.9–98.7%) and 97.0% (95% CI 88.5–99.5%) and specificities of 96.7% (95% CI 91.2–98.9%) and 97.3% (95% CI 91.9–99.3%) relative to herpes simplex virus culture and PCR, respectively. We discuss relevant design characteristics and approaches used for each step of the analytical process to enhance assay sensitivity and provide accurate results in the presence of potential cross-reactive organisms and interfering substances.     

A TaqMan probe-based multiplex real-time PCR method for the specific detection of wild type lumpy skin disease virus with beta-actin as internal amplification control

Lumpy skin disease (LSD) is a transboundary disease of economic importance affecting cattle and buffaloes. In South-Eastern Europe, immunization of cattle with homologous live attenuated vaccines for LSD control has prevented outbreaks since 2017, but has been associated with adverse reactions resembling disease symptoms. Thus, a diagnostic method suitable for disease surveillance in farms during vaccination campaigns with Neethling (Onderstepoort) and SIS type (Lumpyvax) live attenuated LSDV vaccines in Europe should be able to detect the wild type (WT) LSDV in animals with adverse reactions to the vaccines and samples with potentially high titers of the vaccine LSDV. To this end, a real-time PCR method targeting the EEV gene of LSDV was developed for the specific detection of WT strains, along with the use of beta-actin gene as an internal amplification control (IAC). Amplification efficiency of the WT virus target was 99.0% and 98.6%, in the presence and in the absence of high loads of vaccine LSDV, respectively. In the presence of 10^5.6 vaccine LSDV DNA copies, the limit of detection for WT LSDV was 12.6 DNA copies per reaction. The inter-assay CV was 0.04% for WT LSDV and 0.13% for beta-actin. The method can confirm diagnosis in suspect cases irrespective of the presence of the vaccine LSDV DNA by overcoming the masking effect of the WT LSDV. The simultaneous amplification of the beta-actin gene further assures the quality of diagnostic testing. The new method is a surveillance tool, complementing the DIVA real-time PCR during vaccination campaigns and can provide rapid insight on the targeted EEV gene in countries with novel and recombinant LSDV strains.     

Development of a multiplex TaqMan real-time PCR assay for the detection of Chlamydia psittaci and Chlamydia pneumoniae in human clinical specimens

Diagnosis of Chlamydia psittaci and Chlamydia pneumoniae infections has traditionally relied on serological assays. We developed a multiplex real-time PCR assay for detection of C. psittaci, C. pneumoniae and an internal control. Results of this assay demonstrated 100% concordance compared to results of previously tested human clinical specimens.     

双重荧光定量PCR检测西尼罗病毒和基孔肯雅病毒

摘要：目的：建立同时检测西尼罗病毒（WNV）、基孔肯雅病毒（CHIKV）的双重荧光定量PCR法,为临床疑似病例的诊断提供依据。 方法：分别针对WNV CAP基因、CHIKV E1基因保守区设计特异性引物和TaqMan探针,建立并优化双重荧光定量PCR反应体系,评价方法的特异性和灵敏度。 结果：建立的双重荧光定量PCR可同时检测WNV、CHIKV核酸,标准曲线相关系数（r）分别达0.999、0.998,灵敏度达10 copies/μL,具有良好的特异性。 结论：建立了同时检测WNV、CHIKV的双重荧光定量PCR法,但尚需临床进一步验证。     

A real-time PCR assay for detection of emerging infectious Elizabethkingia miricola

Elizabethkingia miricola, a Gram-negative bacillus, is emerging as a life-threatening pathogen in both humans and animals. However, no specific and rapid diagnostic method exists to detect E. miricola. Here, we established a real-time PCR assay for the rapid, sensitive, and specific detection of E. miricola with a wide dynamic range of 10⁸ copies/μL to 10² copies/μL. The detection limit of the real-time assay was 145 copies/μL, which was 100 times more sensitive than conventional PCR. All clinical isolates E. miricola from different host species yield very close Tm (80.25 ± 0.25 °C). Additionally, no cross-reaction or false positives were observed in the assay for non-target bacterial species. The performance of this assay was primarily assessed by testing frog tissue samples. Overall, our study provided a real-time PCR assay, which is a rapid, sensitive, and specific diagnostic method that could be used for early diagnosis and epidemiological investigation of E. miricola.     

Evaluation of the RIDAGENE real-time PCR assay for the detection of GI and GII norovirus

The current study examined the efficacy of the RIDAGENE norovirus (NoV) real-time polymerase chain reaction assay (R-Biopharm, Darmstadt, Germany) for use in a routine diagnostic laboratory. The RIDAGENE assay had an overall sensitivity of 98% but was more sensitive for GII than GI NoV. The assay had a specificity of 98%. The RIDAGENE assay could detect a variety of GI and GII open reading frame 2 genotypes including GI.1, GI.3, GI.8, GI.13, GII.2, GII.3, GII.4 (including the following variants: 2006b, 2009, 2012, and 3 others that have not been assigned), GII.6, GII.12, and GII.13. The assay did not cross react with a number of gastroenteritis viruses including adenovirus, astrovirus, rotavirus, and sapovirus. The assay was straightforward to perform, and for a run of 50 specimens, a result was obtainable in roughly 4 hours. The RIDAGENE assay can be recommended as a valuable detection method for NoV.     

Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3

In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R² > 0.997), and the limits of detection were 37.8 copies/μL, 30.6 copies/μL and 60 copies/μL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.     

基于vvhA基因TaqMan实时荧光定量PCR快速检测创伤弧菌的研究

目的建立基于溶细胞毒素基因vvhA检测创伤弧菌（Vibrio vulnificus）的TaqMan实时荧光定量PCR。方法采用Primer Express软件,设计位于vvhA基因序列保守区的PCR引物和TaqMan探针,建立检测创伤弧菌vvhA基因100bp扩增产物的TaqMan实时荧光定量PCR。采用基因克隆技术,构建作为阳性对照的pMD19-vvhA100重组质粒。以最低扩增循环数（Ct值）、荧光强度增加值（△Rn）为观察指标,对vvhA基因TaqMan实时荧光定量PCR条件进行优化。以不同浓度的创伤弧菌及其它8种细菌DNA和pMD19-vvhA100为模板,对vvhA基因TaqMan实时荧光定量PCR的特异性、敏感性和重复性进行验证。创伤弧菌经腹腔和皮下注射及灌胃感染ICR小鼠,验证vvhA基因TaqMan实时荧光定量PCR对感染小鼠血液、皮下感染组织和肠内容物的检测效果。结果所建立的vvhA基因TaqMan实时荧光定量PCR仅对创伤弧菌DNA或pMD19-vvhA100呈现阳性检测结果,其检测灵敏度可达0．01ng创伤弧菌DNA或10^3个拷贝的pMD19-vvhA100,不同浓度pMD19-vvhA100三次检测结果的SD≤0．79,对腹腔和皮下感染创伤弧菌的小鼠血液、皮下组织标本检测结果均为阳性。结论所建立的创伤弧菌vvhA基因TaqMan实时荧光定量PCR具有快速、稳定、敏感、特异等优点,适合用于临床实验室进行创伤弧菌引起的败血症和创口感染快速诊断。     

Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection

Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species.We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described.The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 10² copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.     

GeneXpert实时荧光定量PCR快速检测艰难梭菌的临床评估

目的对GeneXpert实时荧光定量聚合酶链反应(PCR)在快速检测临床粪便标本中艰难梭菌的应用进行评估。方法采用双拭子蘸取临床未成形粪便标本,一支拭子用于GeneXpert实时荧光定量PCR检测艰难梭菌毒素基因tcdB,另一支用于常规厌氧菌培养检测;对GeneXpert实时荧光定量PCR检测结果与常规厌氧菌培养结果的一致性进行统计学分析,并计算GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值等参数。结果临床收集到141例未成形粪便标本,GeneXpert实时荧光定量PCR检出艰难梭菌毒素基因tcdB阳性42例,其中常规厌氧菌培养阳性34例,两者一致性较好(Kappa=0.775 0,P0.01),GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为87.2%、92.2%、81.0%和94.9%。结论 GeneXpert实时荧光定量PCR直接检测粪便标本中的艰难梭菌具有检测快速、操作简便等优点,有重要的临床应用价值。