One-step RT-PCR Research Articles

A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells

T-cell receptor (TCR) gene therapy is a promising approach for the treatment of infectious diseases and cancers. However, the paired cloning and functional assays of antigen-specific TCRα and TCRβ is time-consuming and laborious. In this study, we developed a novel, rapid and efficient antigen-specific TCR-cloning system by combining three technologies: multiplex one-step RT-PCR, transcriptionally active PCR (TAP) and luciferase reporter assays. Multiplex one-step RT-PCR with leader primers designed from leader peptide sequences of TCRs enabled us to amplify cDNAs of TCRα and β pairs from single T-cells with remarkably high efficiency. The combination of TAP fragments and HEK293T-based NFAT-luciferase reporter cells allowed for a rapid functional assay without the need to construct expression vectors. Using this system, we cloned human TCRs specific for Epstein-Barr virus BRLF-1-derived peptide as well as mouse TCRs specific for melanoma-associated antigen tyrosinase-related protein 2 (TRP-2) within four days. These results suggest that our system provides rapid and efficient cloning of functional antigen-specific human and mouse TCRs and contributes to TCR-based immunotherapy for cancers and infectious diseases.

Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Wild Animals and Ixodidae Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic to central-northeastern China, southern Japan, and the Republic of Korea (ROK). To investigate SFTSV infections, we collected serum samples and ticks from wild animals. Using serum samples and ticks, SFTSV-specific genes were amplified by one-step RT-PCR and nested PCR and sequenced. Indirect immunofluorescence assay (IFA) was performed to analyze virus-specific antibody levels in wild animals. Serum samples were collected from a total of 91 animals: 21 Korean water deer (KWD), 3 Siberian roe deer, 5 gorals, 7 raccoon dogs, 54 wild boars (WBs), and 1 carrion crow. The SFTSV infection rate in wild animals was 3.30% (3 of 91 animals: 1 KWD and 2 WBs). The seropositive rate was 6.59% (6 of 91 animals: 5 KWD and 1 WB). A total of 891 ticks (3 species) were collected from 65 wild animals (9 species). Of the attached tick species, Haemaphysalis longicornis (74.86%) was the most abundant, followed by Haemaphysalis flava (20.20%) and Ixodes nipponensis (4.94%). The average minimum infection rate (MIR) of SFTSV in ticks was 4.98%. The MIRs of H. longicornis, H. flava, and I. nipponensis were 4.51%, 2.22%, and 22.73%, respectively. The MIRs of larvae, nymphs, and adult ticks were 0.68%, 6.88%, and 5.53%, respectively. In addition, the MIRs of fed and unfed ticks were 4.67% and 4.96%, respectively. We detected a low SFTSV infection rate in wild animals, no differences in SFTSV infection rate with respect to bloodsucking in ticks, and SFTSV infection for all developmental stages of ticks. This is the first report describing the detection of SFTSV in wild animals in the ROK.

Identification of Concurrent Infection by Multiple Dengue Virus Serotypes During an Epidemic in 2011 in Pakistan

J Microbiol Exp 2016, 3(3): 00088 DENV-1 and DENV-2 [6]. In 1994 the first lab confirmed cases of DENV-2 was reported from Baluchistan Province [7]. The prevalence of dengue virus serotype 3 in Pakistan was detected during the 2005 dengue virus outbreak in Karachi [8]. The cocirculation of DENV-2 and DENV-3 was identified in 2006 during the outbreak of dengue virus in Karachi [9]. In 2008 the cocirculation of three serotypes DENV-2, DENV-3 and DENV-4 was also documented during the outbreak of dengue virus in Lahore [10]. Recently it is reported that the dominant serotypes of dengue virus circulating in Pakistan from 2007-2009 are DENV2 and DENV-3. The dengue is a major public health problem in Pakistan over the last 20-25 years. In last few years co-circulation of different dengue virus serotypes is identified with mixed infection. From Pakistan only one study in which they reported some cases of mixed infection with DENV-2 and DENV-3.In present study we for the first time reported many cases of mixed infection with DENV-1/DENV-2, DENV-1, DENV-2 and DENV-3 from different geographic areas of Pakistan. The laboratory detection of dengue virus infection is achieved by virus isolation in cell culture, Serologic diagnosis by detection of IgM/IgG antibodies and more sensitive and rapid technique is the Molecular detection. From August to October 2011 a total of 87 serum samples were received at the department of virology National Institute of Health Islamabad Pakistan during an outbreak. These samples were collected from suspected dengue virus cases along the complete history sheet including the date of onset, age; sex etc. All serum samples were stored at -70 until further processing. All serum samples were analyzed for the detection of dengue virus IgM antibodies by using IgM capture ELISA (Panbio Tech Co Brisbane, Australia) according to the instructions provided by the manufacturer. RNA was extracted from 140ul of ELISA Positive serum samples by using QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to manufacturer instructions. In the present study RNA samples were analyzed by the molecular detection by using the real time PCR assay. Real time PCR is a one step assay using Primer pair and Probes specific to dengue serotypes. In single plex reaction mixture 5ul of RNA template was added with 50pmol of each primer and 9pmol of probe in 50ul total iScript one-step RT-PCR kit for Probes. Each reaction tube contains primers and probe for only one dengue serotype. In this assay one RNA sample was tested against four dengue serotypes in four separate reactions tubes [11]. In this technique the use of fluorescent probe enables the detection of PCR reaction product without need of gel electrophoresis. Another advantage of this assay is the ability to determine viral load in the patient sample which is important to understand the severity of dengue infection [12].

Multiplex One-step RT-PCR for Detection and Serotyping of Foot and Mouth Disease Virus in Balochistan

Foot-and-mouth disease (FMD) is a highly infectious and contagious disease of cloven hoofed animals. The disease is endemic in Pakistan caused by Foot-and-mouth disease virus (FMDV) serotype O, A and Asia-1. In order to control FMD in Pakistan, it is essential to have an accurate knowledge of circulating virus strains in the country that will help to select more specific vaccines. Balochistan province (Largest Province area wise) has an important geographical location, having common borders with Afghanistan and Iran that are also FMD endemic countries. Data regarding FMDV strain circulating in this province is scanty. Balochistan being the largest province of the country and having important geographical location needs to be explored for the detection of different serotypes circulating along with their lineages and sub lineages in order to control the disease as well as to access the routes of animal movement in the region, which could be the source of spread of virus. Furthermore, restriction of cross border movement is the need of time, along with the establishment of laboratories having capability for early and accurate diagnosis of the disease. This study suggests that there should be extensive sampling from this province for detection and typing of the virus and if possible, isolation of the circulating strains. This will help to identify Hot Spots in the region for regular monitoring against this alarming threat for the region. The serotype and strain identification will help to improve the current vaccines being used in the region. Sensitive, specific and quick diagnostic tools are needed to control the disease in FMD endemic regions, especially in the areas with limited facilities to diagnose the disease. In this paper, the situation of FMD in Balochistan province of Pakistan has been taken into consideration where FMD is endemic. Balochistan Province has limited diagnostic facilities in terms of infrastructure, equipment, kits, chemicals and reagents to perform all the techniques recommended by OIE, World Organisation for Animal Health, to detect and type the FMD virus. Most of the commercially available assays are too expensive to use for diagnosis with limited resources. This paper describes the optimization of multiplex one-step RT-PCR using specially designed primers for this region. This will serve as a milestone in early detection and serotyping of FMDV in Balochistan.

Multiplex One-step RT-PCR for Detection and Serotyping of Foot and Mouth Disease Virus in Balochistan

Foot-and-mouth disease (FMD) is a highly infectious and contagious disease of cloven hoofed animals. The disease is endemic in Pakistan caused by Foot-and-mouth disease virus (FMDV) serotype O, A and Asia-1. In order to control FMD in Pakistan, it is essential to have an accurate knowledge of circulating virus strains in the country that will help to select more specific vaccines. Balochistan province (Largest Province area wise) has an important geographical location, having common borders with Afghanistan and Iran that are also FMD endemic countries. Data regarding FMDV strain circulating in this province is scanty. Balochistan being the largest province of the country and having important geographical location needs to be explored for the detection of different serotypes circulating along with their lineages and sub lineages in order to control the disease as well as to access the routes of animal movement in the region, which could be the source of spread of virus. Furthermore, restriction of cross border movement is the need of time, along with the establishment of laboratories having capability for early and accurate diagnosis of the disease. This study suggests that there should be extensive sampling from this province for detection and typing of the virus and if possible, isolation of the circulating strains. This will help to identify Hot Spots in the region for regular monitoring against this alarming threat for the region. The serotype and strain identification will help to improve the current vaccines being used in the region. Sensitive, specific and quick diagnostic tools are needed to control the disease in FMD endemic regions, especially in the areas with limited facilities to diagnose the disease. In this paper, the situation of FMD in Balochistan province of Pakistan has been taken into consideration where FMD is endemic. Balochistan Province has limited diagnostic facilities in terms of infrastructure, equipment, kits, chemicals and reagents to perform all the techniques recommended by OIE, World Organisation for Animal Health, to detect and type the FMD virus. Most of the commercially available assays are too expensive to use for diagnosis with limited resources. This paper describes the optimization of multiplex one-step RT-PCR using specially designed primers for this region. This will serve as a milestone in early detection and serotyping of FMDV in Balochistan.

First Molecular Evidence of Citrus yellow vein clearing virus from Citrus in Punjab, Pakistan

Yellow vein clearing disease was first observed on lemon and sour orange trees in Pakistan in 1988 (Catara et al. 1993). It was an important vector-transmitted disease (Onelge et al. 2011) that was then found in India, Turkey, and China. Recently, a putative new member of the genus Mandarivirus, Citrus yellow vein clearing virus (CYVCV), was identified as the putative causal agent of this disease (Loconsole et al. 2012) in Turkey and then in China. However, so far, no data on a virus associated with this disease were available from Pakistan, even though the disease was originally described there. In 2009, a survey was performed to evaluate the occurrence and distribution of citrus viruses and virus-like diseases in Punjab, Pakistan. Field symptoms of distinct yellow vein clearing and leaf distortion were observed on two lemon trees in Faisalabad District. Infected budwood from one symptomatic Eureka lemon (Citrus limon) tree was grafted onto lemons budded on rough lemon rootstock as indicator plants kept in the greenhouse of Citrus Research Institute in China. Six months later, typical yellow vein clearing disease symptoms were visible on lemon seedlings. Total RNA was extracted from symptomatic lemon seedlings using Trizol. One pair of primers, CYV-627F (5′-CAAACAACCAAGCCGCTA-3′) and CYV-1254R (5′-GCCGAACTCTTTCTTTCCG-3′), was used to amplify a ∼600-bp CYVCV specific product by one-step RT-PCR (Invitrogen). The presence of CYVCV was confirmed through sequencing of the 600-bp fragment. Based on the sequences of CYVCV-Turkey, eight primer pairs were designed to amplify overlapping cDNA fragments spanning the CYVCV RNA genome. The 5′ and 3′ termini were determined using the 5′/3′ RACE kit. Each cDNA fragment was cloned with pGEM-T Easy kit and a minimum of five recombinant clones were sequenced. The sequence of the whole genome of CYVCV-Pakistan isolate (KP313241) obtained was 7,529 bp and had 97% identity to the reference sequence from Turkey. CYVCV-Pakistan had the same six open reading frames as CYVCV reported from Turkey. To confirm the virus is still in the field, samples from these two symptomatic trees in Faisalabad District and eight other symptomatic lemon trees with YVC in Sahiwal District were collected in March 2015. RT-PCR tests with primers CYV-627F and CYV-1254R confirmed CYVCV presence in all these samples. To our knowledge, this is the first molecular evidence of CYVCV occurrence in Pakistan, which confirms the strict association of CYVCV infections with lemon trees exhibiting yellow vein clearing disease, as previously reported from Turkey and China. Our finding emphasizes the need for CYVCV indexing in production and distribution of pathogen-free citrus plants in Pakistan.

Epidemiologic characteristics of noroviruses isolated in outpatients with acute gastroenteritis in Hangzhou area, from 2014 to 2015

To explore the epidemiologic characteristics of noroviruses isolated in patients with acute gastroenteritis in Hangzhou between March 2014 and April 2015. Stool specimens and clinical data were collected from 1 109 patients with acute gastroenteritis. Specimens were detected for noroviruses with GⅠand GⅡsubtypes by one-step double real-time RT-PCR. Some of the positive specimens were then randomly selected and amplified by multiplex RT-PCR. Those positive PCR products were sequenced and analyzed phylogenetically for testing the partial capsids of noroviruses. Of the 1 109 stool specimens, positive rate of noroviruses was 26.87% (298/1 109). GⅡgenotype was the major viruses with the proportion as 25.52% (283/1 109), while 1.35% (15/1 109) belonged to GⅠgenotypes. There was no significant difference in the noroviruses detection rate of the different genders (P>0.05). However, in different age groups, GⅡgenotypes were predominant types of noroviruses, and the positive rates of GⅡgenotypes were 16.94% (<5 years-old), 19.45% (5-18 years-old) and 32.26% (≥18 years-old), respectively. In different seasons, noroviruses could be detected all year round, with positive rate as 29.67%-37.08% in the highly epidemic seasons (between December and March of the following year). The distribution trends were seen certain difference between noroviruses-GⅡand GⅠtypes. Additionally, results from the sequence analysis demonstrated that GⅡ-4 genotype was the prevalent strain of GⅡ genotypes, clustered into GⅡ-4/Sydney (46.43%, 13/28) and GⅡ-4/2006b (25.0%, 7/28), while GⅠstrains clustered into GⅠ-1. Noroviruses appeared one of the major pathogens, leading to acute gastroenteritis. G Ⅱgenotypes of noroviruses, especially the G Ⅱ-4/Sydney variant strains and GⅡ-4/2006b variant strains, were considered to be the prevalent strains prevailed in Hangzhou areas from 2014 to 2015.

Whole genome alignment based one-step real-time RT-PCR for universal detection of avian orthoreoviruses of chicken, pheasant and turkey origins

Newly emerging avian orthoreovirus (ARV) variants have been continuously detected in Pennsylvania poultry since 2011. In this paper, we report our recent diagnostic assay development of one-step real-time RT-PCR (rRT-PCR) for the rapid and universal detection of all ARVs or reference strains of chicken, pheasant and turkey origins and six σC genotypes of the newly emerging field ARV variants in Pennsylvania (PA) poultry. Primers and probes for the rRT-PCR were designed from the conserved region of the M1 genome segment 5′ end based on the whole-genome alignment of various ARV strains, including six field variants or novel strains obtained in PA poultry. The detection limit of the newly developed rRT-PCR for ARV was as low as 10 copies/reaction of viral RNA, and 100.50–100.88 tissue culture infectious dose (TCID50)/100μL of viruses. This new rRT-PCR detected all six σC genotypes from the 66 ARV field variant strains and reference strains tested in this study. There were no cross-reactions with other avian viruses. Reproducibility of the assay was confirmed by intra- and inter-assay tests with variability from 0.12% to 2.19%. Sensitivity and specificity of this new rRT-PCR for ARV were achieved at 100% and 88%, respectively, in comparison with virus isolation as the “gold standard” in testing poultry tissue specimen.

Genetic characterization of influenza A (H7N6) virus isolated from a live-bird market in Thailand.

A one-year influenza A virus (IAV) monitoring program was conducted in a live-bird market (LBM) in Thailand. Using one-step real-time RT-PCR (rRT-PCR), 2.39 % of live birds were found to be IAV positive. Twenty viruses could be identified as IAV subtype H7N6. Eight IAV-H7N6 viruses were subjected to whole-genome sequencing and genetic characterization. Phylogenetic analysis showed that the HA gene of Thai H7N6 is grouped with those of the H7 Eurasian viruses. The NA gene is closely related to those of the N6 Eurasian viruses. This is the first report of IAV subtype H7N6 in Thailand.

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.

One-step Quantitative RT-PCR Assays for Detecting, Genotyping and Differentiating Wild-Type Group a Rotaviruses and Vaccine (Rotarix® and RotaTeq®) Strains in Stool Samples.

One-step Quantitative RT-PCR Assays for Detecting, Genotyping and Differentiating Wild-Type Group a Rotaviruses and Vaccine (Rotarix® and RotaTeq®) Strains in Stool Samples.

Evaluation of chikungunya virus infection in children from India during 2009-2010: A cross sectional observational study.

Chikungunya virus, a small (about 60-70 nm diameter), spherical, enveloped, positive, single stranded RNA virus is transmitted by Aedes mosquitoes. After a short period of incubation (3-5 days) symptoms like fever with joint pains and others start appearing. After a gap of 20 years, this virus re-emerged during 2006-2008 in India causing a major outbreak of CHIKV in India. This study was conducted subsequent to the major outbreak in order to evaluate the proportion of chikungunya virus infection in children with suggestive symptoms at three geographical locations of India. Lineage of circulating strains and changes in the E1 structural polypeptide were also determined. Blood samples were collected (in Sodium citrate vacutainer tubes) during 1st June 2009 to 31st May 2010 from children (age 0 ≤ 18 years) suspected to have chikungunya infection, that is, those who presented with sudden onset of fever and/or joint pain, myalgia, and headache from three regions of India, All India Institute of Medical Sciences (AIIMS) in New Delhi, Karnataka Institute of Medical Sciences (KIMS) in Hubli and Sawai Mansingh Medical College (SMS) in Jaipur. Detection of CHIKV antibodies in all acute-phase patient plasma samples was done by IgM ELISA and for samples within ≤5 days of fever, a one-step RT-PCR was carried out on a block thermo-cycler targeting 294 bp region of E1 gene that codes for the viral envelope protein. Comparison of nucleotide and amino acid sequences from few positive samples of two regions was done with African S-27 reference strain using BioEdit. A phylogenetic tree was constructed using MEGA 6 by using the Maximum Likelihood method based on the Kimura 2-parameter model. Out of the 723 acute phase samples tested from three geographical locations of India, Chikungunya virus infection was detected in 249/723 (34.44%) subjects by either IgM Elisa (180/723) or RT-PCR (69/412). RT-PCR was employed in samples collected from children with ≤5 days of fever. Maximum positive cases were from KIMS center, Hubli. Seasonally, positivity varied with number of enrolled cases at KIMS and SMS. Joint pain was significantly associated with CHIKV positivity (P = 0.0156). Presence/absence of certain clinical features varied with age (P < 0.05). Sequence analysis revealed four amino acid changes. Phylogenetic analysis with partial sequences of E1 gene from KIMS (n = 12) and SMS (n = 5) showed that the study isolates clustered with Indian Ocean Lineage strains (IOL) of East, Central and South African (ECSA) type. Evaluation of chikungunya virus infection in children from India during 2009-2010 showed high proportion of CHIKV infection in Southern region of India compared to Northern region. The circulating CHIKV strains were of Indian Ocean Lineage (IOL) group within the East, Central, and South African (ECSA) genotype. However few amino acid changes were observed in E1 polypeptide with reference to African strain S-27 (AF369024). Further studies are needed to know the implications of these changes in vector-pathogen compatibility and host-pathogen interactivity. As a whole, this study highlighted the proportion of CHIKV cases, lineage of causative strain and evolutionary pattern of circulating strain in terms of amino acid changes in the structural protein.

Sensitivity improvement of immunochromatographic strip test for infectious myonecrosis virus detection

An immunochromatographic strip test with enhanced sensitivity for the detection of infectious myonecrosis virus (IMNV) was developed using three monoclonal antibodies specific to N- and C-termini fragments of IMNV capsid protein. This strip test can detect IMNV with 20 times higher sensitivity than the previous test strip and approximately 50 times lower than that of one-step RT-PCR. The shrimp sample can be heat-treated to augment the release of antigen from the muscular tissue and ensure the sterilization of the sample. Due to its high specificity, field friendly and rapid result, this test strip is suitable for surveillance of IMNV outbreaks and confirmation of IMNV infection in shrimp farming. Statement of relevanceThe IMNV strip test will help farmers to monitor IMNV infection during shrimp culture. The test kit has sensitivity of approximately 50 times lower than that of one-step RT-PCR.

The prevalence and genotype diversity of Human Rotavirus A circulating in Thailand, 2011–2014

Human rotavirus A (RVA) is the major infectious virus causing acute watery diarrhea in children, especially those younger than 5years of age, and is a major public health problem in Thailand. Outbreaks of this virus have been reported worldwide. Besides the common genotypes, unusual genotypes providing evidence of inter-species transmission have also been described. Therefore, the aim of this study was to investigate the prevalence and genotypes of RVA in Thailand. A total of 688 samples were collected from children who were hospitalized with acute diarrhea in Chumphae Hospital in Khon Kaen and Chulalongkorn Hospital in Bangkok. RVA was detected using one-step RT-PCR and the genotypes were evaluated by sequencing. Overall, 204 of the 688 samples (30%) were positive for RVA. Nine genotypes were identified: three common in humans (G1P[8] [53%], G2P[4] [18%], G3P[8] [12%]), one feline-like (G3P[9] [1%]), four porcine-like (G4P[6] [0.5%], G5P[6] [0.5%], G9P[8] [0.5%], G12P[6] [1.5%]), and one bovine-like (G8P[8] [13%]). The variation in virus genotypes and the animal-like genotypes detected in this study suggested that a high diversity of RVA types is circulating in the Thai population. Therefore, continuous molecular epidemiological monitoring of RVA is essential and has implications for the national vaccination program.

Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation

Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses’ genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17×103 to 6.56×109 and 3.76×103 to 9.13×1010 genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs.

One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples.

BackgroundDengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.MethodsThe DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.ResultsThe RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 102 to 106 copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.ConclusionsOur results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-1226-z) contains supplementary material, which is available to authorized users.

Rapid Detection and Molecular Typing of Dengue Virus by Using Multiplex-Nested-RT-PCR

world. We have evaluated the combination of one-step RT-PCR and multiplex nested PCR assays for detectingdengue viruses from clinical samples. Twelve patients were screened for the dengue virus, using a pair of primersthat conserve for several Flavivirus. The results showed that in 12 suspect patients, 100% were positive for Flavivirusand there are some genotypic variation among them, that indicated by several RT-PCR products higher than 511 bp,the expected product for RT-PCR. Further assay was performed to clarify the presence and serotypes of dengue virususing multiplex nested PCR. Serotyping results indicated that 83,3% of samples can be confirmed for dengue virus.Among the dengue virus positive 16,7 % are dengue-2, 16.7 % are dengue-3, and the most common 50% are dengue-4,whereas dengue-1 were not found among the patients. The combination of RT-PCR and multiplex nested PCR assaycan be used for rapid analysis dengue samples in early phase which is potentially useful for clinical, epidemiologyand also evolutionary studies.Key words: Flavivirus, dengue virus, serotype, RT-PCR, multiplex nested PCR

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.

Isolation, preparation, and assessment of the immunogenicity effectiveness of a novel inactivated rotavirus vaccine strain: ZTR-68, rotavirus A genotype G1P[8

BackgroundRotavirus is one of the leading causes of diarrhoea in children in both developed and developing countries, resulting in an estimated 600 000 deaths worldwide each year. In China, roughly 13 400 children younger than 5 years die from rotavirus infection each year. We aimed to isolate and characterise a human virus for use as a candidate human inactivated rotavirus vaccine strain. We studied the preparation of the vaccine and assessed its immunogenicity effectiveness. MethodsFrom September, 2009, to November, 2012, we collected 206 stool specimens from children in Yunnan, China, and tested for the presence of rotaviruses by use of enzyme immunoassay and double-stranded RNA-PAGE. G and P typing of the positive samples were done by one-step RT-PCR and sequencing. A new human rotavirus was isolated from a child aged 3 months with acute gastritis from the First People's Hospital of Zhaotong City, Zhaotong, China, and was designated ZTR-68. We did a full genomic analysis of ZTR-68 and tested for identifiable characteristics of the virus. A scalable inactivated rotavirus vaccine was cultured by rolling bottles, purified using low-velocity centrifuge, microfiltration clarification, ultrafiltration concentration, and size-exclusion chromatography, and inactivated by the addition of formaldehyde. Its immunogenicity was studied in Balb/c mice by intramuscular injection. FindingsStrain ZTR-68 was isolated and plaque purified, and its complete genome was sequenced. ZTR-68 is a rotavirus A of genotype G1P[8] and has a close phylogenetic relation to Wa. We confirmed that our rotavirus vaccine was completely inactivated, and rotavirus antigen can be measured by ELISA. After intramuscular injection of the vaccine into BalB/c mice aged 6 weeks, high titres of rotavirus-specific IgG (>20 000) and neutralising activity (roughly 3000) were recovered in the sera of these mice. InterpretationThis newly isolated rotavirus, ZTR-68, is being used to establish a new strain of inactivated rotavirus vaccine. Our findings show that the inactivated rotavirus vaccine has good immunogenicity and provide evidence for further clinical development as an alternative candidate vaccine. FundingThis research was financially supported by the State Project for Essential Drug Research and Development, the Chinese national “Twelfth Five-Year” plan (project number: 2014ZX09102041004); the National High Technology Research and Development Program (“863” Program) of China (project number: 2012AA02A404); and the Yunnan Research Program of Application Foundation and Advanced Technology, self-raised funds (project number: 2013FZ140).

Emergence of quarantine Tobacco ringspot virus in Impatiens walleriana in the Czech Republic

Tobacco ringspot virus (TRSV) is a quarantine pathogen in Europe. During an official inspection in November 2011, Impatiens walleriana plants showing symptoms were found in a nursery in the Czech Republic. The causal agent of the disease was detected as Nepovirus group A by RT-PCR using specific primers of the Nepovirus group. Sequence analysis of PCR fragments confirms that the detected virus is TRSV. TRSV detection in these plants was further confirmed by ELISA and one-step RT-PCR using specific primers. The coat protein (CP) gene of the Czech TRSV isolate was sequenced, and the sequence analysis showed high identity of both nucleotide (99.28%) and amino acid (99.96%) levels compared with other known TRSV isolates from GenBank. Two amino acid motifs characteristic of nepoviruses, FDDY (FDAY) and FWGR (FYGR), were found equally at positions 80 and 497 of the TRSV CP genes, respectively, including the sequences described in this study.