A generic real-time TaqMan assay for specific detection of lapinized Chinese vaccines against classical swine fever

Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N^pro gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.     

Development of a real-time PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus

Summary. A real-time PCR assay based on primer-probe energy transfer (PriProET) was developed to detect swine vesicular disease virus (SVDV). Specificity tests of SVDV and heterologous virus showed specific amplification of SVDV strains only. The amplification plot for the closely related Coxsackievirus B5 remained negative. The sensitivity of assay was five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirmed specific hybridisation of the SVDV strains. Eight of twenty SVDV strains tested, revealed shifted melting points that indicated mutations in the probe region. All predicted mutations were confirmed by nucleotide sequencing. With the PriProET system there is a chance to identify phylogenetically divergent strains of SVDV, which may appear negative in other probe-based real-time PCR assays. At the same time, any difference in melting points may provide an indication of divergence in the probe region. The high sensitivity, specificity, and tolerance toward mutations in the probe region of the SVDV PriProET assay may improve the early and rapid detection of a wide range of SVDV strains, allowing reduced turnaround time and the use of high-throughput, automated technology.     

Development of TaqMan real-time PCR assay for detection and quantitation of reticuloendotheliosis virus

A highly sensitive real-time PCR method was developed in this study for reticuloendotheliosis virus (REV) detection and quantitation. The real-time PCR method, with a minimum detection limit of 10 proviral DNA copies, was 100 times more sensitive than the conventional PCR. It was also shown to be highly specific, as no positive signals were detected for other common avian DNA viruses. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The chicken β-actin gene was co-amplified and used as the internal control to monitor the efficiency of DNA extraction and PCR amplification. Specific pathogen free chickens were infected with REV at different ages and the blood was detected with the real-time PCR method. High levels of proviral DNA were detected in the blood of REV-infected chickens during the experiment and chickens infected early had higher proviral loads from 2 weeks post-infection compared with late infected chickens. This study provides an excellent research and diagnostic tool that can be used for REV detection and quantitation.     

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。     

Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay

Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).     

Development of a Plexor real-time PCR assay for the detection of porcine circovirus type 2

A novel, real-time PCR system for the detection of porcine circovirus type 2 (PCV2) was developed. The system employed Plexor technology and detected 10⁸-10¹ copies per reaction of PCV2 DNA within a recombinant plasmid. The examination of clinical material showed consistent diagnostic sensitivity when samples contained more than 10³ viral copies per reaction. Specificity of Plexor real-time PCR was confirmed using the porcine viruses PCV1, PRRSV, CSFV, TTSuV1 and TTSuV2 employing the melting curve analysis of PCR products. The low values of coefficient of variation in the intra- (1.74%) and inter-assay (2.41%) analysis suggested that the assay was a highly reproducible. The Plexor real-time PCR was compared with three other real-time PCR systems (SYBR Green, TaqMan, LUX) with conclusion that it can be used as a method of choice for the detection and quantitation of PCV2.     

实时RT-PCR检测西尼罗病毒

目的建立西尼罗病毒快速、敏感和特异的实时RT-PCR法,用于西尼罗病毒疾病的早期诊断。方法对Vero细胞增殖的西尼罗病毒分别进行常规RT-PCR和实时RT-PCR法扩增,以PFU／ml为参考标准,比较二者的敏感性和特异性。并用实时RT-PCR法检测西尼罗病毒感染的小鼠组织标本。结果采用所建立的实时RT-PCR法和常规RT-PCR法可分别检测到0．02PFU／ml和2PFU／ml的西尼罗病毒RNA,前者的敏感性比后者高100倍。而对其他黄病毒科成员的检测均为阴性,表明该方法是特异的。采用这种实时RT-PCR法可从西尼罗病毒感染小鼠的血液、脾脏、肾脏和脑组织标本中检测出病毒核酸,且在感染后第2天最先自血液和脾脏中检测到病毒,在感染后1周的脑组织中的病毒滴度最高。结论本研究建立的实时RT-PCR法具有很高的敏感性和特异性,因此该方法可用于西尼罗病毒疾病的早期监测和流行病学研究。     

Evaluation of a BK virus real-time PCR assay designed using novel bioinformatics technology

Monitoring of active polyomavirus BK (BKV) infections by quantitative real-time PCR is becoming a progressively more routine practice in the care of renal transplant patients due to the potential for these infections to injure transplanted kidneys. Quantitative BKV results from a previously validated, laboratory-developed real-time PCR assay based on commercially available MGB Alert® reagents were compared to results obtained from the same urine and plasma specimens using a commercially designed real-time PCR assay by IntelligentMDx. When compared qualitatively, the two assays performed identically with the exception of one urine specimen in which BKV DNA was detected near the lower limit of quantification by the MGB Alert® assay. A quantitative comparison of the results showed an average 0.55 log₁₀ copies/mL difference between the two assays. These findings suggest that despite small differences, the IntelligentMDx assay could be adopted for clinical BKV monitoring of renal transplant patients.     

Molecular beacon real-time PCR detection of swine viruses

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 × 10¹ copies of target and are linear between 2 × 10⁹ and 2 × 10² copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.     

Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus

The design and development of a 5′ conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2 × 10² copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2 × 10² to 2 × 10⁸ copies/μl. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease.     

A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus

Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex™ RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0 × 10¹ and 6.0 × 10² copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV.     

Development and comparison of a Primer-Probe Energy Transfer based assay and a 5' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5′ conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2 × 10⁸ to 2 × 10² copies/μl. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5′ conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV.     

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(?) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas.     

Development of a real-time PCR assay for rapid identification of methicillin-resistant Staphylococcus aureus from clinical samples

A major drawback of mecA PCR to detect methicillin-resistant Staphylococcus aureus (MRSA) directly from patient materials is the high frequency of methicillin-resistant coagulase-negative staphylococci. Therefore, a reliable detection method for MRSA from clinical samples using real-time PCR was developed. The PCR assay targeting the integration site (orfX) of the staphylococcal cassette chromosome mec (SCCmec) was evaluated in MRSA SCCmec reference strains (n = 9), MRSA ST strains (n = 16) and clinical isolates of MRSA (n = 124), MSSA (n = 53), methicillin-resistant coagulase-negative staphylococci (n = 47), and methicillin-susceptible, coagulase-negative staphylococci (n = 32). The diagnostic values of the assay were 98% sensitivity and 100% specificity. Furthermore, the PCR detection method was evaluated with 60 swabs from different body sites which were incubated overnight in brain-heart infusion. The PCR gave positive results for 27 of 29 swabs which were found to contain MRSA by conventional methods. The diagnostic values of the PCR assay for these samples were 93% sensitivity and 100% specificity. To determine the in vitro sensitivity of the assay, swabs were inoculated with serially diluted bacterial suspensions. After overnight enrichment the detection limit of the PCR was less than 10 CFU/swab. This new real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory.     

猪瘟基因疫苗免疫小鼠体液及细胞免疫动态变化研究

目的：探讨猪瘟基因疫苗免疫小鼠后机体产生体液和细胞免疫发生规律，为猪瘟基因疫苗推广应用提供科学依据。方法：应用FACS、MTT法及间接ELISA试验对猪瘟基因疫苗免疫小鼠脾脏及外周血中CD4^ 和CD8^ 淋巴细胞数、淋巴细胞的转化功能及特异的抗猪瘟病毒血清IgG抗体水平等动态变化进行了观察。结果：猪瘟基因疫苗免疫小鼠后脾细胞及外周血淋巴细胞对ConA和LPS均有明显的反应性。CD4^ 和CD8^ 细胞数量在免疫后20天开始反应，32天达到高峰后开始下降。鼠血清特异性猪瘟病毒血清抗体IgG随免疫时间延长而增加。结论：猪瘟基因疫苗免疫动物可诱导机体体液及细胞免疫。