One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples.
ABSTRACT: Porcine cytomegalovirus (PCMV) is a pathogen that must be removed from pigs for use as organ donors in xenotransplantation. Recently, it has been found that when donor pigs are infected with PCMV, a pig-to-non-human-primate xenotransplantation lower transplant survival by 2-3 times. Therefore, highly sensitive methods are needed to maintain designated pathogen free (DPF) pig and screen for xenografts. The purpose of this study was to evaluate the performance of commercially available method with one-tube nested real-time PCR assay to quickly detect PCMV infection in clinical samples and compare the results with those of sequence analysis. Molecular diagnostic methods were used to evaluate 127 samples, including tissues and blood samples from pigs suspected of PCMV infection. The detection rate for positive PCMV was 38.6% (n = 49), 23.6% (n = 30), and 12.6% (n = 16) in one-tube nested real-time PCR, nested PCR, and conventional PCR methods, respectively. All PCMV-positive samples in conventional PCR or nested PCR methods were also positive in the one-tube nested real-time PCR assay. All the PCR products in the three methods were checked for amplification of PCMV gene by PCR and subsequent direct sequencing. The results of one-tube nested real-time PCR were found to be consistent with those of sequence analysis for all the samples and showed good agreement (? = 1). Our study found that the one-tube nested real-time PCR assay is more sensitive than the other two methods. This assay required approximately 1.5 h for completion. Therefore, we concluded that one-tube nested real-time PCR assay is a fast and reliable method for the characterizing pathogen responsible for PCMV infection.
Project description:The porcine cytomegalovirus (PCMV) is a herpesvirus that may pose a risk for xenotransplantation using pig cells, tissues, or organs. Here, three orthotopic pig heart transplantations into baboons were studied. To detect PCMV, a real-time PCR and a Western blot assay based on four PCMV protein sequences, including two tegument proteins, were used. The transmission of PCMV from the donor pig to the recipient baboon was found in two cases, despite PCMV not being detected in the blood of the donor pigs by real-time PCR. Although it was not in the blood, PCMV was detected in different organs of the donor pigs, and in sibling animals. Immunohistochemistry using an antiserum that is specific for PCMV detected virus protein-expressing cells in all of the organs of the recipient baboon, most likely representing disseminated pig cells. Therefore, for the first time, the distribution of PCMV in organs of the donor pigs and the recipient baboons was described. In addition, baboon cytomegalovirus (BaCMV) was found activated in the recipient, and a screening for hepatitis E virus (HEV) and porcine lymphotropic herpesviruses (PLHV) was performed. For the first time, a cross-reactivity between antibodies directed against PCMV and BaCMV was found.
Project description:Porcine cytomegalovirus (PCMV) infection is widely prevalent among pigs, and PCMV is one of the viruses which may be transmitted during xenotransplantation using pig cells, tissues, or organs. While human cytomegalovirus (HCMV) is a major risk factor for allotransplantation, it is still unclear whether PCMV is able to infect human cells or pose a risk for xenotransplantation. Previously, it was shown that transmission of PCMV after pig kidney to non-human primate transplantations resulted in a significantly reduced survival time of the transplanted organ. To detect PCMV, PCR-based and immunological methods were used. Screening of pigs by Western blot analyses using recombinant viral proteins revealed up to 100% of the tested animals to be infected. When the same method was applied to screen human sera for PCMV-reactive antibodies, positive Western blot results were obtained in butchers and workers in the meat industry as well as in normal blood donors. To exclude an infection of humans with PCMV, the sera were further investigated. PCMV is closely related to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and a sequence alignment of glycoprotein B suggests that the antibodies may cross-react with identical epitope sequences. HCMV is not related with PCMV, and no correlation between antibody reactivity against PCMV and HCMV was detected. These data indicate that antibodies against PCMV found in humans are cross-reactive antibodies against HHV-6.
Project description:This study described a TaqMan based real-time fluorescent quantitative PCR (qPCR) method to detect porcine cytomegalovirus (PCMV) infection, targeting the conserved region of the DNA polymerase (DPOL) gene. The standard curve showed a linear regression relationship with a coefficient of 0.999 and a slope of y?=?-3.249x?+?38.958 corresponding to the amplification efficiency at 99.8%. The limit of the qPCR method was 51.9?copies/?l. The established qPCR method showed excellent specificity, with no cross-reaction observed with common porcine pathogens. The coefficient of variation for intra-assay and interassay variability ranged up to 1.51% and 2.24%, respectively. PCMV positive signals can be found in semen using this qPCR method, which suggested that we should pay more attention to PCMV contamination in semen in order to eliminate PCMV infection in artificial insemination and xenotransplantation.
Project description:Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNF? were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.
Project description:This is the first published report of a PCR assay for detecting porcine cytomegalovirus (PCMV), the causative agent of inclusion body rhinitis in pigs. The DNA to be tested was extracted directly from lungs and nasal scrapings of pigs with various clinical syndromes. Fifty-nine percent (74 of 126) of tested pigs with various clinical syndromes were found to be PCR positive for PCMV. It is hoped that veterinary diagnostic laboratories will benefit by using this PCR assay for routine testing and surveillance of PCMV in pigs.
Project description:Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.
Project description:BACKGROUND:Respiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment. RESULTS:A locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay. CONCLUSION:mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.
Project description:Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or alpha-1,3-galactosyltransferase gene knockout miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell microchimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.
Project description:In clinical situations where a diagnostic real-time PCR assay is not sensitive enough, leading to low or falsely negative results, or where detection earlier in a disease progression would benefit the patient, an unbiased pre-amplification prior to the real-time PCR could be beneficial. In Amp-PCR, an unbiased random Phi29 pre-amplification is combined with a specific real-time PCR reaction. The two reactions are separated physically by a wax-layer (AmpliWax®) and are run in sequel in the same sealed tube. Amp-PCR can increase the specific PCR signal at least 100×10(6)-fold and make it possible to detect positive samples normally under the detection limit of the specific real-time PCR. The risk of contamination is eliminated and Amp-PCR could replace nested-PCR in situations where increased sensitivity is needed e.g. in routine PCR diagnostic analysis. We show Amp-PCR to work on clinical samples containing circular and linear viral dsDNA genomes, but can work well on DNA of any origin, both from non-cellular (virus) and cellular sources (bacteria, archae, eukaryotes).
Project description:We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 10(3) to 10(11) copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.