SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of dengue virus type 3 infections

Dengue is the most important arthropod-borne viral disease in the world. A rapid diagnostic test for dengue is warranted, and real-time polymerase chain reaction may improve diagnosis. TaqMan and Sybr Green systems were evaluated for the diagnosis of dengue virus type 3 (DENV-3) infections. Out of 77 patients with clinical suspicion of dengue infection, specific IgM antibodies were detected in 40 patients. DENV-3 was detected and quantitated in 17 IgM-positive samples by both systems. These assays were shown to be rapid, and specific for detection of DENV-3, and that for diagnostic purposes, there is no difference between these two assays.     

Clinical evaluation of the NS1 antigen‐capture ELISA for early diagnosis of dengue virus infection in Brazil

The fact that the diagnosis of infection with dengue virus is usually made by detecting IgM antibodies during the convalescent phase of the disease interferes with disease management and, consequently, with reducing mortality rates. This study evaluated the sensitivity and specificity of detection of NS1 in samples of patients suspected of acute dengue virus infection in Brazil. The results were used to institute treatment and the sensitivity and specificity of detection of NS1 were compared to the results of detection of IgM, virus isolation, and RT‐PCR. Detection of NS1 yielded better results than RT‐PCR and virus isolation. When considering IgM detection and RT‐PCR positive results as “gold standards,” the sensitivity and specificity of the NS1 assay were 95.9% and 81.1%, respectively. All patients enrolled in the study were treated promptly and had an uneventful course of the disease. The detection of NS1 provided better results than the diagnostic techniques used currently during the acute phase of disease (RT‐PCR and virus isolation). Detection of NS1 is an important tool for the diagnosis of acute dengue infection, particularly in highly endemic areas, allowing for rapid treatment of patients and reduction of disease burden. J. Med. Virol. 82:1400–1405, 2010. © 2010 Wiley‐Liss, Inc.     

Comparison of real-time fluorescent RT-PCR and conventional RT-PCR for the detection of hepatitis E virus genotypes prevalent in China

To compare the specificity and sensitivity of a real-time fluorescent RT-PCR assay with conventional RT-PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non-A-C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real-time RT-PCR could detect the same final dilution of genotype 1 as conventional RT-PCR but could detect a 10-fold lower concentration of genotype 4 than conventional RT-PCR. Of 416 samples from patients with a clinical diagnosis of non-A-C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real-time and conventional RT-PCR, respectively. The concordance of real-time and conventional RT-PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti-HEV IgM by real-time and conventional RT-PCR, respectively, and 31 and 26 of 245 samples negative for anti-HEV IgM, were positive by real-time and conventional RT-PCR, respectively. All amplicons positive by conventional RT-PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real-time RT-PCR assay is more sensitive than conventional RT-PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China.     

Improved diagnosis on a daily basis of enterovirus meningitis using a one-step real-time RT-PCR assay

The detection of the enterovirus genome in cerebrospinal fluid (CSF) by PCR techniques has proved to be more sensitive than traditional cell culture for the diagnosis of enterovirus meningitis. However, PCR assays are time consuming and labor intensive, particularly if separate hybridization steps are used to confirm the specificity of positive findings. The aim of this study was to develop a one-step real-time RT-PCR assay with LightCycler (LC) technology that was sensitive, rapid, and easy to perform in routine practice. The enterovirus detection limit was determined by testing 10-fold limiting dilution series of cell culture stocks with the echovirus 25 (E-25) prototype strain and with the third European Union Quality Control Concerted Action (EU-QCCA) enterovirus proficiency panel. A total of 100 CSF specimens were investigated in a comparative study. With the E-25 strain, the detection limit of the real-time assay was 286 TCID50/ml (50% tissue culture infective dose). When samples of the EU-QCCA panel were tested, our assay gave identical results (detection limit down to 3.6 TCID50/ml) to those of the reference laboratory, which used one-step RT-PCR assay. When CSF specimens were tested, there was a correlation between the real-time assay and the conventional in-house assay in 96 of 100 CSFs tested. This one-step real-time assay allows rapid enterovirus detection in CSF since results are obtained in 3 hr as against 36 hr with the "in-house" RT-PCR assay. This new assay is now being used in routine practice, and allows diagnosis on a daily basis.     

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.     

Enhancement of detection and quantification of rotavirus in stool using a modified real-time RT-PCR assay

A sensitive and specific real-time RT-PCR assay to detect rotavirus in stool samples was optimized and validated using a wide range of rotavirus genotypes. The target of the original TaqMan(R) assay is an 87 bp fragment of the highly conserved non-structural protein 3 (NSP3) gene. Here we modified the original assay by introducing degeneracy into the forward primer to account for sequence variation between rotavirus genotypes, added four nucleotides at the 3' end of the reverse primer to reduce its stability, and modified the probe label. Amplification and detection conditions were optimized using purified dsRNA from two cultivated strains. The limit of detection of the modified assay was calculated to be approximately 44 genome copies per reaction. To validate the reactivity of the assay, 103 archived RNAs that had been extracted from stools and genotyped during routine U.S. surveillance were tested. Samples were selected to represent both rare and common genotypes that have been detected in U.S. children. Nine genotypes known to be circulating in the United States were detected by the real-time assay demonstrating broad reactivity. In addition, other enteric viruses were not detected demonstrating that the assay is specific for rotavirus and does not cross-react with other viruses potentially present in stool samples. This real-time assay is an important addition to the arsenal of molecular tools available to quickly identify rotavirus in stool samples during routine surveillance.     

登革热病毒多重荧光PCR检测及基因分型方法的研究

目的建立一种登革热病毒双靶基因多重荧光PCR检测方法,用于登革热病毒的实验室诊断和基因分型。方法选取登革热病毒Ⅰ-Ⅳ型病毒保守区设计型特异性引物探针和通用型引物探针。评估多重荧光PCR检测方法的特异性、重复性和检测限;并对20份阳性样本进行检测。结果20个登革热阳性核酸标本在通用型检测全部为阳性,特异性型别检测发现登革热病毒Ⅰ型10例、登革热病毒Ⅱ型3例、登革热病毒Ⅲ型3例、登革热病毒Ⅳ型4例;20名正常无症状人群标本提取的核酸和HIV、HCV和HEV通用型和特异性型别检测全部为阴性。梯度检测的变异系数均小于5％。对登革热Ⅰ-Ⅳ型病毒检测最低检测限达10^3 eopies／ml。结论本研究建立的登革热病毒双靶基因多重荧光PCR检测及分型方法具有特异性好、重复性好、快速易操作等优点,可用于登革热病毒的快速检测和基因分型鉴定。     

Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma

Dengue is mosquito-borne virus infection that annually causes ∼50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3′ end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.     

Comparison of loop-mediated isothermal amplification, real-time PCR, and virus isolation for the detection of herpes simplex virus in genital lesions

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.     

Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue virus infection

Purpose: This study was undertaken to evaluate the efficacy of NS1 antigen (Ag) assay as an early marker for dengue virus (DV) infection. Materials and Methods: Group I evaluated the performance of NS1 antigen (Ag) assay in comparison to MAC-ELISA and their detection rate when performed together in a single sample. Six hundred acute/early convalescent sera were screened by both the assays. Group II evaluated the efficacy of a single assay in 30 acute phase sera of paediatric OPD patients screened only by NS1 Ag assay. Group III evaluated the specificity of NS1 assay in comparison to MAC-ELISA on 40 samples included as controls. Results: In Group I, 140 (23.3%) and 235 (39.1%) samples were positive by NS1 assay and MAC-ELISA respectively. The detection rate increased to 320 (53.3%) when both the assays were used together on a single sample. NS1 Ag positivity varied from 71.42% to 28.4% in acute and early convalescent sera, conversely IgM detection rate was 93.61% and 6.38% in early convalescent and acute phase sera respectively (P < 0.0001). In Group II, 66.66% (20) samples were positive by NS1 assay. All the samples in Group III were negative showing 100% specificity of both the assays. Conclusion: NS1 Ag assay holds promise in early diagnosis of dengue infection. When used in combination with MAC-ELISA on a single sample it significantly improves the diagnostic algorithm without the requirement of paired sera.     

Diagnosis of Oropouche virus infection by RT-nested-PCR.

Using the RT-PCR with primers that anneal to the 5' and the 3' extremities of the genome segments of bunyaviruses and internal primers that anneal to the S segment of Simbu serogroup viruses in a nested PCR it was possible to amplify the Oropouche virus (ORO) genome from the sera of three patients. These results show that this RT-nested-PCR is a useful tool for rapid diagnosis of Oropouche fever infections.     

应用长链RT-PCR法扩增我国登革2、4型病毒株全长cDNA

目的：采用长链RT-PCR技术扩增登革2型及4型病毒基因组全长cDNA，为构建登革病毒全长cDNA克隆、表达，深入阐明致病机理及探索新型疫苗奠定基础。方法：根据已测定的登革2、4型病毒全基因组序列，设计上下游引物。从感染登革病毒的乳鼠脑中提取病毒基因组RNA，采用长链RT-PCR技术进行扩增。为检验扩增产物的特异性，以PCR产物为模板扩增覆盖基因组的10个片段。将含有复杂二级结构的5′非编码区扩增片段，在377A型自动测序仪进行序列分析。结果：扩增出登革2、4型病毒基因组全长近11kb cDNA分子，非编码区测序结果表明扩增产物为登革2、4型病毒所特有。结论：利用长链RT-PCR首次成功扩增出登革病毒全长cDNA分子。