One-step Multiplex Real-time RT-PCR Research Articles

TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease

Effective control and monitoring the spread of foot-and-mouth disease (FMD) relies upon rapid and accurate laboratory detection of FMD virus (FMDV). Therefore, in this report, a multiplex TaqMan probe-based one-step RT-qPCR assay simultaneously targeting FMDV 5′UTR and 3Dpol regions, and 18S rRNA housekeeping gene (as an internal control) in a single reaction tube was developed and evaluated. The multiplex one-step RT-qPCR assay specifically detected viral genome in both FMDV-infected cell culture suspensions and clinical samples collected from known-FMD infected animals. The assay could detect FMDV RNA in the archived FMDV cell culture isolates (n = 120) collected during the last two decades in India. In addition, the new assay could also detect viral RNA in the FMD suspected clinical samples (n = 740) collected from various field outbreaks. At a cut-off Ct-value of <38, the assay could detect at least 20 and 10 copies of FMDV 3Dpol and 5′UTR genes, respectively. Further, the multiplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.3% to 3.03% for FMDV-3Dpol gene target, and from 1.44% to 4.69% for 5′UTR gene target. In addition, it was found that the new assay could be used to detect viral genome in a variety of samples (epithelium, saliva, OPF, milk and blood) without any significance difference in the detection limit of the assay. Hence, the multiplex one-step RT-qPCR assay could be considered a valuable tool for the detection of FMDV in India.

One-step Multiplex Real-time RT-PCR for Molecular Detection and Typing of Dengue Virus Infection From Paraffin-embedded Tissues During the Brazilian 2019 Outbreak.

Formalin-fixed paraffin-embedded (FFPE) tissues are an important source for investigation of dengue virus (DENV) infection, particularly when blood or fresh frozen (FF) samples are unavailable. Histopathologic features and immunohistochemistry may have poor sensitivity and serotype determination is not always possible. Viral RNA genome detection tests are faster and considered the most sensitive technique for this kind of analysis, however, the use of molecular methods applied to FFPE tissues is still limited. The authors applied a single-step multiplex reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for the investigation of DENV infection and typing to FFPE samples of 32 fatal cases received during the 2019 outbreak that occurred in São Paulo state, Brazil. The authors compared the results with those obtained using FF tissues. Of the 24 cases with both FF and FFPE samples, 22 (91.67%) of the FF and 19 (76.20%) of the FFPE specimens were positive. Two cases (8.33%) tested negative in both types of samples. All 8 cases with only FFPE samples available were positive. The accuracy (87.5%) of the RT-qPCR for DENV in FFPE samples were satisfactory. Although the cycle quantification (Cq) values were significantly higher in these materials (P<0.0001, Wilcoxon signed-rank test) when compared with FF tissues, Spearman's rank coefficient indicated a good correlation between the Cq values from both sample types (P=0.0063; rho=0.576). RT-qPCR applied to FFPE samples improved detection of DENV in fatal cases and represents a useful tool for diagnosis and epidemiologic studies.

A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2

Early diagnosis of SARS-CoV-2 infected patients is essential to control the dynamics of the COVID-19 pandemic. We develop a rapid and accurate one-step multiplex TaqMan probe-based real-time RT-PCR assay, along with a computational tool to systematically analyse the data. Our assay could detect to a limit of 15 copies of SARS-CoV-2 transcripts—based on experiments performed by spiking total human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an internal control reference gene with dilutions ranging from 1-100 ng for human RNA on a cohort of 26 clinical samples. 5 of 26 patients were confirmed to be infected with SARS-CoV-2, while 21 tested negative, consistent with the standards. The accuracy of the assay was found to be 100% sensitive and 100% specific based on the 26 clinical samples that need to be further verified using a large number of clinical samples. In summary, we present a rapid, easy to implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface (GUI) to analyze the raw qRT-PCR data in an unbiased manner at a cost of under $3 per reaction and turnaround time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.

Phylogenetic analysis of influenza B viruses in Qingdao from 2011 to 2018

Objective To investigate the molecular epidemiological characteristics of hemagglutinin (HA) and neuraminidase (NA) of influenza B viruses (IBV) isolated in Qingdao from 2011 to 2018. Methods A total of 12 236 samples of influenza-like cases in Qingdao from 2011 to 2018 were collected to extract viral RNAs. All samples were screened for influenza A viruses (IAV) and IBV by one-step multiplex real-time RT-PCR. Lineages of IBV were identified. One hundred and eighty-two strains of IBV were selected to amplify HA and NA genes by RT-PCR and then analyzed by sequencing. Phylogenetic analysis and variation analysis of genes and amino acids were carried out. Results IBV was detected almost every year in Qingdao from 2011 to 2018. The positive rate was only slightly lower than that of IAV (4.99% vs 6.21%). B/Victoria linkage had two prominent epidemic years (2011-2012, 2015-2016), while B/Yamagata linkage had three (2013-2014, 2014-2015, 2017-2018). Most of the infected people were children under 10 years old, and the people infected with the two lineages had similar age characteristics. Phylogenetic analysis of HA genes showed clusters in Victoria clades of 1A and 1B and Yamagata clades of 2 and 3. IBV of Yamagata lineage had more amino acid mutation sites than those of Victoria lineage in HA genes with greater genetic diversity. The B/Yamagata strains had 12 amino acid mutations and the B/Victoria strains had seven in four major epitopes. In the receptor binding sites, two amino acid mutations were detected in the B/Yamagata strains and three in the B/Victoria strains. In Qingdao, 26 strains of IBV were intra-lineage reassortments, mostly of the B/Victoria lineage, and 23 strains were inter-lineage reassortments, mostly between HA-B/Yamagata and NA-B/Victoria strains. A possible resistant strain to NA inhibitor was found. Conclusions The significance of IBV in seasonal influenza should not be neglected. Amino acid substitution, insertion/deletion and gene reassortment were the main strategies for the natural evolution of IBV. Influenza surveillance was of great importance and influenza vaccine strains needed to be updated in time. Key words: Influenza B virus; Hemagglutinin (HA) gene; Neuraminidase (NA) gene; Phylogenetic analysis

Performance evaluation of gastrointestinal viral ELIte panel multiplex RT-PCR assay for the diagnosis of rotavirus, adenovirus and astrovirus infection

Rotavirus, adenovirus, norovirus and astrovirus are considered to be among the major causes of sporadic cases and outbreaks of acute gastroenteritis globally. Rapid and accurate identification of enteric viruses is still a challenge for the clinical laboratory. Recently, several molecular platforms for the detection of viral enteric pathogens have become available. In this study, the diagnostic accuracy of InGenius Gastrointestinal Viral (GV) Elite Panel, a newly developed one-step multiplex real-time RT-PCR assay simultaneously detecting rotavirus, adenovirus and astrovirus, was evaluated retrospectively analyzing an archival collection of 128 stool samples of children hospitalized with acute gastroenteritis. The overall sensitivity and specificity for the GV assay was 100% and 96.2% for rotavirus, 96.9% and 100% for astrovirus, 100% and 100% for adenovirus, respectively. The InGenius GV assay showed a high concordance with the reference methods and was able to detect all tested genotypes of rotavirus (including G1, G3, G4, G9 and G12P[8] and G2P[4]), adenovirus and astrovirus (AstV-1 and 2). Studies of considerable sample size are required to determine robust Cycle threshold cut-off values to effectively correlate infection to disease. These preliminary results suggest that InGenius GV assay can be recommended as a valuable method for accurate diagnosis of epidemic and sporadic gastroenteritis.

Multiplex real-time RT-PCR for detection and distinction of Spondweni and Zika virus

Zika (ZIKV) and Spondweni viruses (SPOV) are closely related mosquito borne flaviviruses in the Spondweni serogroup. The co-circulation and similar disease presentation following ZIKV and SPOV infection necessitates the development of a diagnostic tool for their simultaneous detection and distinction. We developed a one-step multiplex real-time RT-PCR (ZIKSPOV) to detect and distinguish between SPOV and ZIKV by utilizing a single primer set combined with virus specific hydrolysis probes. The ZIKSPOV assay was compared to published virus specific real-time RT-PCR assays and the limit of detection was comparable. The SPOV reference strain AR94 was detectable to 0.001 TCID50 per PCR reaction, while African lineage ZIKV (MR 766) was detectable to 0.002 TCID50 per reaction and Asian lineage ZIKV (H/PF/2013) to 0.05 TCID50 per reaction. The ZIKSPOV assay did not detect other flaviviruses, indicative of its specificity for Spondweni serogroup. The ZIKSPOV assay is a useful addition to arbovirus diagnostic and surveillance tools in areas where ZIKV and SPOV are expected to co-circulate. Further evaluation is required to demonstrate the application of the assay for detection of ZIKV and SPOV in mosquito and human clinical samples.

A one-step multiplex real-time RT-PCR for the universal detection of all currently known CCHFV genotypes

Crimean-Congo hemorrhagic fever (CCHF) is a fatal disease in humans, which is endemic in many countries of Africa, Southern Asia and Southeastern Europe. It is caused by the Crimean-Congo hemorrhagic fever virus (CCHFV), which is an arthropod-borne virus (arbovirus) transmitted by ixodid ticks, mainly of the genus Hyalomma. Animals like hares, hedgehogs, cattle, camels and small ruminants can become infected without developing clinical signs. Seroconversion occurs after a short viremia of up to two weeks, and thus seroprevalence studies in ruminants can be used to reveal risk areas for the human population. Virus detection by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is essential to prove an actual circulation of CCHFV in a country and is also used as diagnostic method for acute human CCHFV infections. In this study, a new universal one-step multiplex real-time RT-qPCR for the sensitive and specific detection of CCHFV is presented. For this purpose, 14 new primers and 2 probes were simultaneously used to detect RNAs representing all six CCHFV genotypes. Additionally, a GC-mirrored sequence within the synthetic RNAs enables the discrimination between true positive samples and unintentional laboratory contaminations. CCHFV negative samples from different animal species and ten different members of the order Bunyavirales were eventually tested to reveal the specificity of the new RT-qPCR.

Simultaneous detection of respiratory syncytial virus and human metapneumovirus by one-step multiplex real-time RT-PCR in patients with respiratory symptoms

BackgroundBoth respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance.MethodsIn order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay.ResultsOur one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R2 > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 102-109copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously.ConclusionsIn summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

Development and Evaluation of a SYBR Green–Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses

Chikungunya virus (CHIKV) and dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. In this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate DENV serotypes 1, 2, 3, and 4 and CHIKV. This SYBR Green I–based one-step multiplex real-time RT-PCR assay is highly sensitive and specific for CHIKV and DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV and to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, and 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- and CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, and 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of DENV and CHIKV in clinical samples.

Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene

BackgroundDengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy. ObjectivesThis study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches. Study designSerotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens. ResultsThe LOD of the assay was 3.0 log10 RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8–47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection. ConclusionsThe assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.

Molecular epidemiology and phylogenetic analyses of influenza B virus in Thailand during 2010 to 2014.

Influenza B virus remains a major contributor to the seasonal influenza outbreak and its prevalence has increased worldwide. We investigated the epidemiology and analyzed the full genome sequences of influenza B virus strains in Thailand between 2010 and 2014. Samples from the upper respiratory tract were collected from patients diagnosed with influenza like-illness. All samples were screened for influenza A/B viruses by one-step multiplex real-time RT-PCR. The whole genome of 53 influenza B isolates were amplified, sequenced, and analyzed. From 14,418 respiratory samples collected during 2010 to 2014, a total of 3,050 tested positive for influenza virus. Approximately 3.27% (471/14,418) were influenza B virus samples. Fifty three isolates of influenza B virus were randomly chosen for detailed whole genome analysis. Phylogenetic analysis of the HA gene showed clusters in Victoria clades 1A, 1B, 3, 5 and Yamagata clades 2 and 3. Both B/Victoria and B/Yamagata lineages were found to co-circulate during this time. The NA sequences of all isolates belonged to lineage II and consisted of viruses from both HA Victoria and Yamagata lineages, reflecting possible reassortment of the HA and NA genes. No significant changes were seen in the NA protein. The phylogenetic trees generated through the analysis of the PB1 and PB2 genes closely resembled that of the HA gene, while trees generated from the analysis of the PA, NP, and M genes showed similar topology. The NS gene exhibited the pattern of genetic reassortment distinct from those of the PA, NP or M genes. Thus, antigenic drift and genetic reassortment among the influenza B virus strains were observed in the isolates examined. Our findings indicate that the co-circulation of two distinct lineages of influenza B viruses and the limitation of cross-protection of the current vaccine formulation provide support for quadrivalent influenza vaccine in this region.

Development and evaluation of multiplex real-time RT-PCR assays for seasonal, pandemic A/H1pdm09 and avian A/H5 influenza viruses detection

Since the pandemic influenza A (H1N1) 2009 ((H1N1)pdm09) virus spread all over the world, the (H1N1)pdm09 virus has been circulating with seasonal influenza viruses. We developed rapid and sensitive one-step multiplex real-time RT-PCR assays (rRT-PCR) for simultaneous detection of influenza viruses currently circulating in humans, and the avian A/H5 virus. The detection limit of each assay was 4.8 to 1 copies per reaction and no cross-reactivity with other major respiratory pathogens was found. Analytical positive predictive value (PPV), negative predictive value (NPV) sensitivity and specificity were 100%, 94.1%, 93.7% and 100%, respectively. Clinical evaluation revealed that 1,976 (16.5%) of 11,963 throat swabs from patients with respiratory symptoms were confirmed as 1,651 (83.6%) A/H1pdm09, 308 (15.6%) A/H3 and 17 (0.8%) B virus during the 2010–2011 influenza season. Collectively, the multiplex rRT-PCR assays described here provide a practical tool for reliable implementation of influenza surveillance and diagnosis.

One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

BackgroundDetection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD.ResultsA mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT).ConclusionsThe mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

Increased Peripheral T Cell Responses to EBV-Infected Cells with Frequent Detection of EBV-DNA In Plasma and Viral mRNA In Peripheral B-Cells In Immunocompetent EBV-Positive Diffuse Large B-Cell Lymphoma Patients

AbstractAbstract 4163 Introduction:It is well known that immunocompromised patients(pts) such as post-transplantation or HIV infection are at increased risk of EBV-associated B-cell lymphoproliferaitive disorders. However, the immunological status of immunocompetent EBV-positive diffuse large B-cell Lymphoma (DLBCL) has not been well elucidated. For example, EBV-positive DLBCL in the elderly is defined as an EBV-positive clonal B-cell lymphoid proliferation occurring in pts more than 50 years of age and without any known immunodeficiency in WHO classification 2008, accounting for 5 – 10% of DLBCL in Japan. This disease is speculated to be related to the immunological deterioration accompanying the aging process. We conducted a multi-center prospective study to assess EBV status and the T cell response to EBV of peripheral blood (PB) in EBV-positive DLBCL in comparison with EBV-negative DLBCL pts and normal old-healthy persons (old-HPs). Patients and Methods:Fifteen newly-diagnosed pts with EBV-positive DLBCL, 8 pts with EBV-negative DLBCL, and 16 old-HPs were enrolled. Median ages of pts with EBV-positive DLBCL, pts with EBV-negative DLBCL, and old-HPs were 74.5 years (range 29–82 years), 71 years (48-79), and 65 years (60-74), respectively. Among the 15 pts with EBV-positive DLBCL, 2 were pyothorax-associated lymphoma, 2 were treated with methotrexate for rheumatoid arthritis, and the remaining 11 pts were without past histories predisposing to immunodeficiency. The diagnosis of EBV-positive DLBCL was made by positive signals for in situ hybridization using EBV-encoded small RNA (EBER) on paraffin section. To analyze T cell reactivity to autologous EBV-infected B-cell lymphoblastoid cell lines (LCL), CFSE-labeled peripheral blood mononuclear cells (PBMCs) were co-cultured with irradiated autologous LCL, and the division indices (DI) of CD4+ and CD8+ T cells were determined on day 5 (Cytometry 34:143, 1998). Percentage of proliferating CFSElow IFN-γ+CD4+ T cells and CFSElow IFN-γ+CD8+ T cells was assessed on day 7. EBV DNA load in PBMCs and plasma was determined by quantitative real-time PCR. CD19+ B cells were separated from of PBMCs by immunomagnetic sorting, and viral mRNA expression of B cells was quantified by one-step multiplex real-time RT-PCR. Results:(1) Plasma EBV-DNA was detectable in 13 of the 15 EBV-positive DLBCL pts but in none of the 8 EBV-negative DLBCL pts or the 16 old-HPs. Copy number of EBV-positive DLBCL was significantly higher than EBV-negative DLBCL (p<0.00001) or old-HPs (p=0.0001). EBV-DNA in PBMC was positive in 10 of the 15 EBV-positive DLBCL pts, 2 of 8 EBV-negative DLBCL pts, and 2 of 15 old-HPs. (2) EBER1 of PB B cells was detected in all 10 EBV-positive DLBCL pts, 4 of 7 EBV-negative DLBCL pts, and 7 of 14 old-HPs. BamHI A rightward transcripts (BARTs) of B-cells was detected in 8 of 10 EBV-positive DLBCL pts, 3 of 10 EBV-negative DLBCL pts, and 2 of 14 old-HPs. Latent membrane protein 2 (LMP2) of B cells was detected in 2 of 10 EBV-positive DLBCL pts, 2 of 14 old-HPs, and none of 5 EBV-negative DLBCL pts. LMP1 of B cells was detected in 1 of 10 EBV-positive DLBCL pts, none of 7 EBV-negative DLBCL pts, and none of 14 old-HPs. EBV-encoded nuclear antigen 1 (EBNA1) or EBNA2 were not detected in any of the pts nor old-HPs. (3) DI of CD4+ T cells for 5 days in EBV-positive DLBCL (median 1.41) was significantly higher than in old-HPs (median 0.53) (p=0.0009), and DI of CD8+ T cells of EBV-positive DLBCL (median 1.94) showed a higher tendency than old-HP (median 1.35). (4) Percentage of CFSE-low IFN-γ+CD4+ T cells in EBV-positive DLBCL (median 9.8%) was significantly higher than in old-HPs (median 2.2%) (p=0.002), and percentage of CFSE-low IFN-γ+CD8+ T cells in EBV-positive DLBCL (median 12.0%) was significantly higher than in old-HPs (median 6.6%)(p=0.025). Conclusions:Measurement of the plasma EBV-DNA copy number is a good indicator for the diagnosis of EBV-positive DLBCL, and the frequent detection of EBV viral mRNA of peripheral B-cells in EBV-positive DLBCL pts might be associated with greater viral load. Proliferative and INF-γ secreting responses of both peripheral CD4+ T cells and CD8+ T cells to EBV-infected cells were increased in EBV-positive DLBCL compared to old-HPs, which might be driven by an elevated viral load. These findings suggest that systemic immunological reaction to EBV might be intact in EBV-positive DLBCL. Disclosures:No relevant conflicts of interest to declare.

Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays

In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capabe of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6–H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10 3 copies/μl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.

Tissue Factor Gene Expression Analysis in Circulating Monocytes*

Background: There is growing evidence that an increasein stimulated and tissue factor(TF)-expressing monocytesdrive the coagulation system to a hypercoagulablestate. In order to further investigate this phenomenon,methods are needed that allow sensitive, reliable andspecific quantification of TF-expressing monocytes. Materialsand Methods: Two procedures of one-step multiplexreal-time RT-PCR were developed that allow absolutequantification of TF, CD14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA transcripts.In order to minimize preanalytical changes of the mRNAprofile, a blood sampling system which is based on integratedRNA stabilization was used. Results: An in vitrolipopolysaccharide (LPS) model was used for initialassay evaluation. LPS-induced TF gene expression patternswere similar to previously described findings. Withthe use of the whole blood RNA stabilization system, themarker transcripts were preserved for up to 24 h at roomtemperature and for up to 5 days at 4 °C. Monocytic TFtranscription analysis in different patient groups identifiedsignificant higher levels in thrombophilic patientsthan in a representative control population. In all patientsand control probands analyzed we found significantlyhigher levels of TF transcripts in carriers of the G allele ofthe TF-603A/G promotor polymorphism. Conclusion: Thenewly developed RT-PCR allows sensitive, reliable andspecific quantification of TF transcripts in whole bloodsamples.