Multiplex Reverse Transcription Polymerase Chain Research Articles

Prevalence and characterization of rotaviruses in children hospitalized for diarrheal disease in a tertiary care hospital, Pune

Background: Diarrhoea remains the second most common cause of death among children below 5 years globally. Among various enteric pathogens, rotavirus appears to be the most important aetiological agent of acute gastroenteritis in infants and young children. Increased understanding of epidemiology of rotavirus infections is needed to improve the vaccine efficacy. Aim: This study aims to determine prevalence rotavirus infection and prevalent circulating strains of rotavirus in and around Pune. Setting and Design: Prospective hospital-based study. The study was approved by Institutional Ethical Committee. Materialsand Methods: Stool samples (n = 100) were collected from children aged <5 years, hospitalised for acute diarrhoea in paediatric ward at a tertiary care hospital. Samples were subjected for rotavirus antigen capture ELISA. The viral RNA was subjected to multiplex reverse transcription polymerase chain reaction to amplify VP7 genotypes G1–G4, G8–G10 and G12 and VP4 genotypes P[4], P[6], P[8], P[9], P[10] and P[11]. Nontypable rotavirus strains were sequenced. Results: About 35% stool samples were positive for rotavirus antigen by ELISA. G9P[4] (28.6%) was found to be the most prevalent rotavirus strain. The detection of emerging strain G12P[6] (14.3%) and rare reassortant strain G9P[4] was the significant finding. Conclusion: Genotypes found in circulation are not present in the currently used vaccine. Thus, an emergence of newer genotypes over a period calls for the continued surveillance and genomic characterisation of rotaviruses to improve the vaccine efficacy.

Prevalence of Rotavirus in Under 5 Year Old Children Hospitalized for Acute Gastroenteritis, Swaziland, 2013 - 2014

<i>Background:</i> Rotavirus is the most common cause of severe diarrhoea among children < 5 years of age worldwide. To determine the prevalence of severe rotavirus infection in children admitted with acute gastroenteritis attending Mbabane Government Referral Hospital in the Hhohho region and Raleigh Fitkin Memorial (RFM) Hospital in the Manzini Region of Swaziland, sentinel surveillance was conducted in January 2013 – December 2014. <i>Methods:</i> This is hospital based sentinel surveillance study. A total of 331 children under the age of five years with acute diarrhoea that were admitted to the hospitals were enrolled into the sentinel surveillance and 302 cases had stool samples tested for rotavirus antigens by enzyme immunoassay ProSpec T Rotavirus kit. The G and P genotypes were established by multiplex nested reverse transcription polymerase chain reaction. <i>Results:</i> Rotavirus was detected in 159 (52.6%) of the 302 children with acute diarrhoea that had stool specimens collected and 108 (68%) of positive cases of rotavirus gastroenteritis were between 6-11 months of age and 91% were below the age of two years. Rotavirus infection occurred with peaks between June - August. During the 2013 – 2014 period, 146 samples were subjected for reverse transcription polymerase chain reaction and genotyping assays. The most common genotypes detected in Swaziland were 82% G2P [4] in 2013 and 93% G1P [8] in 2014. <i>Conclusion:</i> Swaziland would benefit by introducing rotavirus vaccine and hence reduce the hospitalization burden of managing acute diarrhoea cases attributed to Rotavirus. The data from the sentinel hospitals can also be used to monitor the effectiveness of the vaccine once introduced. 1. National Reference Laboratory, Ministry of health, Swaziland- MOH 2. World Health Organization, Inter country support team for East and Southern Africa, Zimbabwe, Harare, 3. Ministry of health, Swaziland 4. World Health Organization, Swaziland 5. Mbabane Government Hospital, Swaziland 6. Raleigh Fitkin Memorial Hospital, Swaziland

Kinase-Activating Fusions in Pediatric High-Risk B-Lineage Acute Lymphoblastic Leukemia (ALL): a Report from the Dana-Farber Cancer Institute (DFCI) ALL Consortium

Abstract Background. Recurrent chromosomal rearrangements carry prognostic significance in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). Recent genome-wide analyses identified a diverse spectrum of chromosomal rearrangements resulting in novel chimeric fusions associated with poor prognosis when treated with conventional chemotherapy. These fusions are observed more frequently in NCI High-Risk (HR) B-ALL compared with NCI Standard Risk (SR) patients. They often activate ABL and JAK-STAT signaling pathways and have demonstrated sensitivity to the relevant tyrosine kinase inhibitors (TKIs) in in vitro assays and ex vivomodels. The objective of this study was to determine the frequency of NCI HR B-ALL patients enrolled on DFCI ALL Consortium Protocol 05-001 with a kinase-activating fusion that would be amenable to TKI therapy and to describe their associated clinical characteristics and outcomes. Methods. Between 2005-2011, 219 NCI HR, Philadelphia chromosome (Ph)-negative, B-ALL patients were enrolled on DFCI ALL Consortium Protocol 05-001, 105 of whom had sufficient material to undergo kinase fusion testing by validated multiplex reverse transcription polymerase chain reaction (RT-PCR) assays. A total of 35 kinase fusions of ABL-class (ABL1, ABL2, PDGFRB, CSF1R), JAK2 and CRLF2 rearrangements were examined. IGH@-CRLF2 and EPOR rearrangements were not assessed. Fusion products were predicted by NCBI BLAST algorithms, confirmed by singleplex PCR and Sanger sequencing and aligned using CLC Main Workbench Version 7.6.1. IKZF1 deletion (del) status had previously been assessed by multiplex ligation-dependent probe amplification (MLPA). Fisher's exact test and the Wilcoxon rank sum test were used to compare patient characteristics to those with and without any identified fusion for categorical and continuous variables respectively. Event-free survival (EFS) and overall survival (OS) were estimated with the Kaplan-Meier method and compared using a log rank test. Univariate and multivariable Cox proportional hazards models of EFS were constructed. Results. Among 105 NCI HR, Ph-negative, B-ALL patients, 16 (15%) were found to harbor an ABL-class fusion (ETV6-ABL1: n=1; FOXP1-ABL1: n=1; SFPQ-ABL1: n=1; ZC3HAV1-ABL2: n=1) or a fusion activating the JAK-STAT pathway (P2RY8-CRLF2: n=8; PAX5-JAK2: n=4). Sixty-nine percent of patients with an identified fusion (Fusion +) had a concomitant IKZF1 del (n=11). Features associated with fusion-positivity were age of 10 years or older (p=0.003), male sex (p=0.03), Hispanic ethnicity (p=0.01) and IKZF1 del (p=0.0005) (Table 1). Fifty percent of Fusion+ patients experienced an event (induction death (n=1); induction failure (n=1); or relapse (n=6)) compared to 24% of patients without a fusion. The 5-year EFS and OS were 48% (95% CI 22-70%) and 68% (95% CI 39-85%) for Fusion+ patients compared to 78% (95% CI 67-85%) and 88% (95% CI 79-93%) for those without fusions (Figure 1). In univariate analysis, fusion-positivity (HR: 2.66, p=0.02) and IKZF1 del (HR: 3.21; p=0.0018) were each significantly associated with inferior EFS, while age and presenting leukocyte count were not. In multivariable analysis, IKZF1 del, but not fusion-positivity, retained statistical significance (HR: 2.64, p=0.02). Conclusion. Fifteen percent of NCI HR, Ph-negative, B-ALL patients enrolled on DFCI ALL Consortium 05-001 were found to have a kinase-activating fusion. Fusion+ patients frequently harbored concomitant IKZF1 deletion and had an inferior outcome. Future studies should focus on developing clinical strategies to rapidly identify these patients at diagnosis and to test whether the addition of the relevant TKIs to their treatment will improve their outcome. Disclosures Asselin: Jazz Pharmaceuticals: Consultancy, Speakers Bureau; Sigma Tau Pharamceuticals: Consultancy. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Assessment of Molecular Response in CML Patients with Atypical BCR-ABL Tanscripts. Recommendations By the EUTOS Collaboration

▪Approximately 2‐3% of CML patients (pts) have variant BCR‐ABL fusions transcripts that cannot be monitored by RQ‐PCR using standard methodologies. Most rare variants are accounted for by seven variant fusions (e1a2/3, b2/3a3, e6a2, e8a2, e19a2). Within the European Treatment and Outcome Study (EUTOS) we sought to establish and validate robust RQ-PCR methods for each of these abnormalities. RNA was extracted from leukocytes obtained from 20 mL peripheral blood. Samples were received either locally or by mail and spent between 1 to 3 days in transit. Multiplex reverse transcription polymerase chain reaction (RT-PCR) experiments were performed to identify BCR-ABL1 transcripts (Cross et al., Leukemia 1994). In all cases, gel electrophoresis showed an unusual band. PCR products were then sequenced to characterize BCR-ABL1 rearrangements. Monitoring of residual disease was carried out by RQ-PCR with specific forward and/or reverse primers and probes using a serial dilution of individual BCR-ABL1 or GUSB plasmid DNA calibrators. GUSB transcripts were quantified as internal control and results were expressed as the BCR-ABL1/GUSB ratio with no conversion according to the international scale (IS) as it is recommended for only typical major (M) BCR-ABL1 and cannot be applied to other transcripts in CML. 43 pts from 8 prospective studies and pts outside of studies were investigated. A total of 435 samples (1-72 per patient, median 8) were analyzed. Pts expressed 6 different atypical BCR-ABL1 transcripts (e19a2, n=18; e1a2, n=10; b2a3, n=5; b3a3, n=5; b2a3&b3a3, n=2, e8a2, n=3). At the start of the observation period, ratios BCR-ABL1/GUSB ranged between 0.01% and 179.1% (median 13.75%). Follow-up results were determined in comparison to the starting level and expressed as relative log reduction. 9 pts were in deep molecular remission considering a minimum GUSB transcript level of 20,000/reaction. During the observation period, six pts relapsed with a significant increase of BCR-ABL1/GUSB ratios. We conclude that very few pts are diagnosed with unusual BCR-ABL1 transcripts, but their characterization is important for proper assessment of treatment response in affected patients, and to avoid false negative assessment of residual disease status. Based on the individual PCR strategies, results cannot be expressed with precision on the International Scale and thus the common molecular milestones and triggers for treatment discontinuation are difficult to apply. We suggest that residual disease in these cases should be assessed primarily by considering trends in BCR-ABL/reference gene ratios over time. DisclosuresSaussele:NOVARTIS PHARMA: Consultancy, Honoraria; BMS: Honoraria. Cross:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Development and application of a universal and simplified multiplex RT-PCR assay to detect five potato viruses

Potato virus X (PVX), Potato virus Y (PVY), Potato leafroll virus (PLRV), Potato virus M (PVM), and Potato virus A (PVA) commonly cause mixed infections and major economic losses in potato crops, so a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay has been greatly needed to detect all five viruses simultaneously. Previously published primers were evaluated, and five pairs of highly specific primers were selected for use. The quintuplex RT-PCR assay was optimized for primer concentration, annealing temperature, number of PCR cycles, and extension time. Finally, the quintuplex RT-PCR assay was successfully established. The amplified target fragments of 711 base pairs (bp) for PVX, 447 bp for PVY, 336 bp for PLRV, 520 bp for PVM and 273 bp for PVA, and the viral origins of the PCR products were confirmed by sequencing. This system can be easily customized for simplex, duplex, triplex, tetraplex, and quintuplex RT-PCRs by changing only the primer pairs while keeping the final reaction volume and the concentrations of other reagents constant to detect any combination of PVX, PVY, PLRV, PVM, and PVA viruses. In this way, the detection systems can be customized to the characteristics of viral infections specific to China’s potato-producing regions for excellent detection efficiency and meeting the needs of potato producers. The detection limit of this assay was up to 10−3 dilution in tetraplex and quintuplex RT-PCR, and even higher in simplex, duplex, and triplex RT-PCR. The efficiency of the quintuplex RT-PCR was compared with that of a DAS-enzyme-linked immunosorbent assay to detect the five viruses in 320 field potato samples, 195 in vitro plantlets and pooled potato samples. This new multiplex RT-PCR method is not only effective but also faster, cheaper, more sensitive, and more reliable than DAS-ELISA. It is especially suitable for the testing of in vitro plantlets and pooled samples of postharvest seed tubers.

Potential causative agents of acute gastroenteritis in households with preschool children: prevalence, risk factors, clinical relevance and household transmission.

Acute gastroenteritis (AGE) morbidity remains high amongst preschool children, posing a significant societal burden. Empirical data on AGE-causing agents is needed to gauge their clinical relevance and identify agent-specific targets for control. We assessed the prevalence, risk factors and association with symptoms for enteropathogens in households with preschool children. A monthly-repeated cross-sectional survey of enteropathogens in households with preschool children was performed. A parent-child pair per household (n = 907 households) provided faecal samples and reported their symptoms and potential risk exposures. Samples were tested by multiplex reverse transcription polymerase chain reaction (RT-PCR) for 19 enteropathogens. Associations were assessed using logistic regression. 28.3% of children (n = 981) and 15.6% of parents (n = 971) carried pathogenic bacteria and/or Escherichia coli-associated pathogenicity genes, and 6.5% and 3.3% carried viruses, respectively. Giardia lamblia (4.6% of children, 2.5% of parents) and Dientamoeba fragilis (36%, 39%, respectively) were the main parasites, and were associated with pet exposure. Living in rural areas was associated with carriage of pathogenic E. coli, norovirus I and D. fragilis. Pathogenic E. coli was associated with summertime and livestock exposure. Attending day-care centres increased the risk of carrying norovirus, sapovirus and G. lamblia. Viruses occurred mainly in winter and were associated with AGE symptoms. Child-parent associations were found for bacterial pathogenicity genes, viruses, G. lamblia and D. fragilis. Enteropathogens spread widely in households with preschool children, particularly viruses, which more often cause symptoms. While bacteria predominate during summer and in those exposed to livestock, viruses predominate in wintertime and, like G. lamblia, are widespread amongst day-care centre attendees.

Detection and typing of human-infecting influenza viruses in China by using a multiplex DNA biochip assay

Rapid identification of the infections of specific subtypes of influenza viruses is critical for patient treatment and pandemic control. Here we report the application of multiplex reverse transcription polymerase chain reaction (RT-PCR) coupled with membrane-based DNA biochip to the detection and discrimination of the type (A and B) and subtype (human H1N1, human H3N2, avian H5N1 and avian H7N9) of influenza viruses in circulation in China. A multiplex one-step RT-PCR assay was designed to simultaneously amplify the HA and NA genes of the four subtypes of influenza A viruses and NS genes to discriminate type A and B viruses. PCR products were analyzed by a membrane-based biochip. The analytical sensitivity of the assay was determined at a range of 2–100 copies/reactions for each of the gene transcripts. Eighty one clinical samples, containing 66 positive samples with evident seasonal influenza virus infections, were tested, which gives the clinical sensitivity and specificity of 95.5% and 100% respectively. For the avian influenza samples, 3 out of 4 H5N1 samples and 2 out of 2 H7N9 avian samples were correctly identified. We argue this method could allow a rapid, reliable and inexpensive detection and differentiation of human-infecting influenza viruses.

Anti-inflammatory effect of dialysable leucocyte extract in a rat model of osteoarthritis: histopathological and molecular characterization

Objectives: To evaluate the effect of dialysable leucocyte extract (DLE) on pro- and anti-inflammatory profiles in a rat model of osteoarthritis (OA).Method: Forty-eight male Wistar rats were divided into three groups: normal rats without treatment, OA rats treated with placebo, and OA rats treated with DLE. After treatment, the animals were killed to obtain cartilage for histological analysis and to determine the expression of pro- and anti-inflammatory cytokines by reverse transcription multiplex polymerase chain reaction (RT-MPCR) and immunohistofluorescence analyses.Results: Histological analysis revealed that OA cartilage from rats treated with DLE displayed similar characteristics to non-OA cartilage from the control group. The OA cartilage treated with placebo showed alterations in the cellular architecture and in chondrocyte cluster formation. Analysis of cytokine expression by RT-MPCR showed that OA cartilage from DLE-treated rats expressed platelet-derived growth factor (PDGF), interferon (IFN)-γ, and fibroblast growth factor (FGF)-2, similar to non-OA cartilage from the control group. However, OA cartilage from rats treated with placebo expressed interleukin (IL)-1, PDGF, and I kappa B (IκB). Confocal immunodetection of FGF-2, PDGF, and non-phosphorylated IκB showed that they were distributed in the cytoplasm of most chondrocytes in OA cartilage from DLE-treated rats whereas no nuclear factor kappa B (NF-κB) expression was observed in the nuclei. Instead, in OA cartilage from the placebo group, only weak FGF-2 staining was observed, PDGF and IκB were not detected, and NF-κB was strongly observed in both cytoplasm and nuclei.Conclusions: Our findings suggest that DLE treatment modifies the OA process, promoting the expression of anti-inflammatory cytokines and diminishing the inflammatory effects, avoiding the nuclear translocation of NF-κB in chondrocytes.

Recent viral pathogen in acute gastroenteritis: a retrospective study at a tertiary hospital for 1 year.

PurposeViral gastroenteritis among children is mainly caused by rotavirus, norovirus, astrovirus, or adenovirus strains. However, changing socioeconomic conditions and a rotavirus vaccination program may be affecting the prevalence of these viral infections. Therefore, we aimed to elucidate the season-specific trends in viral infections for facilitating prophylaxis and surveillance in our region. MethodsWe evaluated 345 pediatric patients (203 males, 142 females; age, 1 month to 16 years) who visited the CHA Bundang Medical Center because of gastroenteric symptoms between June 2014 and May 2015. The specimens were simultaneously tested for norovirus, rotavirus, astrovirus, and adenovirus via multiplex reverse transcription polymerase chain reaction. Clinical characteristics of patients were analyzed retrospectively.ResultsThe most common virus was norovirus, followed by rotavirus, adenovirus, and astrovirus. Of all viral infections, 45.2% occurred mainly between 6 and 24 months of age; in particular, norovirus infection mostly occurred in all age groups except those below 6 months of age, when rotavirus was most prevalent. In addition, seasonal variation was observed, such as norovirus infection from December to February, rotavirus infection from February to April, and adenovirus infection from July to October.ConclusionOur results showed that the most common cause of acute pediatric viral gastroenteritis had changed from rotavirus to norovirus in our patients, because of effective rotaviral vaccination. We recommend the management of food and personal hygiene in accordance with age or seasons as well as active vaccination for preventing viral gastroenteritis.

Prevalence of Abelson murine leukemia viral oncogene homolog-breakpoint cluster region fusions and correlation with peripheral blood parameters in chronic myelogenous leukemia patients in Lorestan Province, Iran.

Context:Chronic myelogenous leukemia (CML) is a chronic malignancy of myeloid linage associated with a significant increase in granulocytes in bone marrow and peripheral blood. CML diagnosis is based on detection of Philadelphia chromosome and “Abelson murine leukemia viral oncogene homolog” (ABL)-“breakpoint cluster region protein” fusions (ABL-BCR fusions).Aims:In this study, patients with CML morphology were studied according to ABL-BCR fusions and the relationship between the fusions and peripheral blood cell changes was examined.Materials and Methods:All patients suspected to chronic myeloproliferative disorders in Lorestan Province visiting subspecialist hematology clinics who were confirmed by oncologist were studied over a period of 5 years. After completing basic data questionnaire, blood samples were obtained with informed consent from the patients. Blood cell count and morphology were investigated and RNA was extracted from blood samples. cDNA was synthesized from RNA and ABL-BCR fusions including b3a2 and b2a2 (protein 210 kd or p210), e1a2 (protein 190 kdor p190), and e19a2 (protein 230 kdor p230) were studied by multiplex reverse transcription polymerase chain reaction method. Coexistence of e1a2 and b2a2 (p210/p190) fusions was also studied. The prevalence of mutations and their correlation with the blood parameters were statistically analyzed.Results:Of 58 patients positive for ABL-BCR fusion, 18 (30.5%) had b2a2 fusion, 37 (62.71%) had b3a2 fusion and three (3.08%) had e1a2 fusion. Coexistence of e1a2 and b2a2 (p210/p190) was not observed. There was no significant correlation between ABL-BCR fusions and white blood cell count, platelet count, and hemoglobin concentration.Conclusions:The ABL-BCR fusions in Lorestan Province were similar to other studies in Iran, and b3a2 fusion had the highest prevalence in the studied patients studied.

Viruses causing severe acute respiratory infections (SARI) in children ≤5 years of age at a tertiary care hospital in Rajasthan, India.

Background & objectives:Severe acute respiratory infection (SARI) is one of the leading causes of death among children worldwide. As different respiratory viruses exhibit similar symptoms, simultaneous detection of these viruses in a single reaction mixture can save time and cost. The present study was done in a tertiary care children's hospital for rapid identification of viruses causing SARI among children less than or equal to five years of age using multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) kit.Methods:A total of 155 throat swabs were collected from equal number of children suspected to have SARI and processed for extraction of nucleic acids using automated extraction system. Multiplex real-time RT-PCR was done to identify the viruses in the samples.Results:The overall positivity for viruses in the study was found to be 72.9 per cent with a co-infection rate of 19.5 per cent. Human metapneumovirus (HMPV) was the predominant virus detected in 25.7 per cent children followed by influenza A (H1N1)pdm09, human rhinovirus (HRV) and human adenovirus (HAdV) in 19.9, 11.0 and 8.8 per cent children, respectively. The HMPV was at its peak in February 2013, HAdV showed two peaks in March-April, 2012 and November 2012-March 2013 while HRV was detected throughout the year.Interpretation & conclusions:Multiplex real-time PCR helped in rapid identification of viruses. Seventeen viruses were detected in SARI cases with overall positivity of 72.9 per cent. HMPV was the most predominant virus. However, for better clinico-virological correlation, studies are required with complete work up of all the aetiological agents, clinical profile of patients and treatment outcome.

MULTIPLEX REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION FOR CHICKEN TUMOR VIRUS DETECTION

Tumor diseases occur frequently in chickens causing a great economic loss. Infected chickens’ pathological lesions are not pathognomonic. This study developed an accurate diagnosis for tumor diseases in chickens. Specific primers to reticuloendotheliosis virus (REV), avian leucosis virus subgroup A (ALV-A), avian leucosis virus subgroup J (ALV-J), and Marek’s disease virus (MDV) were combined into one tube with a single step multiplex reverse transcription polymerase chain reaction (mRT-PCR) performed to amplify the genes from each virus. A total of 117 sample pools containing blood and tissues were collected from chickens. Three of these pools (2.6%) showed REV positive, 22 (18.8%) ALV-J positive, 1 (0.8%) ALV-A positive, and 3 (2.6%) MDV positive. A total of 268 blood samples were used to compare the viral RNA detection from plasma using RT-PCR and provirus DNA from buffy coat using PCR. The result showed no difference from both tests. In conclusion, the present mRT-PCR could be used for tumor virus detections in chickens.

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.

Molecular epidemiology of enteric viruses and genotyping of rotavirus A, adenovirus and astrovirus among children under 5 years old in Gabon

This study aimed to determine the prevalence of enteric viruses causing gastroenteritis, and the circulating stains, in Gabonese children under five years old who visited health centers between March 2010 and June 2011. Stool specimens were collected and sent for analysis to CIRMF (Centre International de Recherches Médicales de Franceville). Stools were screened for six enteric viruses (rotavirus, adenovirus, norovirus I and II, sapovirus, human astrovirus) by means of a multiplex real-time reverse transcription polymerase chain reaction, and Rotavirus A, Adenovirus and Astrovirus were genotyped. Among the 317 specimens analyzed, 193 (60.9%) were positive for at least one enteric virus. Rotavirus A (RVA) (27.1%) was the most frequently detected virus, followed by human Adenovirus (HAdV) (19.6%), Norovirus II (NoVs-II) (13.9%), Norovirus I (NoVs-I) (9.1%), Sapovirus (SaV) (9.5%) and human Astrovirus (HAstV) (6.3%). One-third of the 193 positive samples contained more than one virus. The most common Rotavirus A genotype was G6P[6]. Various HAdV serotypes were found. HAstV-1 was identified. These findings improve our knowledge of circulating enteric viruses in Gabon. The emergence of unusual G6P[6] strain of rotavirus A, predominant, suggested a particular epidemiological surveillance of circulating rotavirus strains during the introduction of vaccination in Gabon.

A multiplex RT-PCR for simultaneous detection and identification of five viruses and two viroids infecting chrysanthemum

Pathogens causing significant economic losses in chrysanthemum include tomato aspermy virus (TAV), chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd). A multiplex reverse transcription polymerase chain reaction (RT-PCR) method, using specific primer sets for each virus or viroid, was developed for simultaneous detection and differentiation of TAV, CVB, CMV, TMV, PVY, CChMVd, and CSVd. The RT-PCR method was validated by testing chrysanthemum samples collected from different regions of China. In this study, CVB, TAV, TMV, PVY, CSVd, CMV, and CChMVd were detected, respectively, in 24.7 %, 17.5 %, 4.4 %, 4.4 %, 2.9 %, 2.5 %, and 1.5 % of the samples tested. These results indicate that CVB and TAV (24.7 % and 17.5 %) are common, whereas CMV, TMV, CChMVd, CSVd, and PVY (all below 5 %) are less frequently encountered. This new multiplex RT-PCR method has potential to be used routinely in large-scale virus and viroid surveys.

Bead-based suspension array for simultaneous differential detection of five major swine viruses.

A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/μL (corresponding to 0.5-6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.

Isolation & molecular characterization of human parainfluenza virus in Chennai, India.

Background & objectives:Human parainfluenza virus (HPIV) accounts for a significant proportion of lower respiratory tract infections in children as well as adults. This study was done to detect the presence of different subtypes of HPIV from patients having influenza like illness (ILI).Methods:Throat and nasal swabs from 232 patients with ILI who were negative for influenza viruses were tested by multiplex reverse transcription polymerase chain reaction(mRT-PCR) for the detection of human parainfluenza virus. All samples were inoculated in rhesus monkey kidney (LLC-MK2) cell line.Results:Of the 232 samples, 26(11.2%) were positive by mRT-PCR and nine (34.6%) showed cytopathic effect with syncytium formation for HPIV and all were HPIV-3 serotype, other serotypes like 1,2,4 were negative. The HPIV-3 strains (HN gene) were sequenced and analysed. Two novel mutations were identified at amino acid residues 295 and 297.Interpretation & conclusions:The mRT-PCR assay offers a rapid, sensitive and accurate diagnostic method for detection of HPIV which enables early detection and control. In our study there was a predominance of HPIV among 1-5 yr age group and the school going age group was less affected. Further studies need to be done to characterize HPIV isolated from different parts of the country.

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10−5 for viroids and 10−4 for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.

A multiplex RT-PCR assay for simultaneous detection of four viruses from sweet cherry

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection of four viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus-2 (LChV-2) and Cherry green ring mottle virus (CGRMV). Random hexamer primer was used for cDNA synthesis. Detection primers were designed against the genomic sequences of four viruses. The reaction conditions were firstly optimized by selecting primers combinations and standardizing the individual primer proportion and PCR protocols. Then, sensitivity of multiplex RT-PCR was evaluated. The assay could detect all four viruses in diluted cDNA (10−6, about 8.64×10−4ngμL−1) and RNA (10−1, about 0.2μg). The reliability of the assay was evaluated by cloning and sequence alignments. The developed multiplex RT-PCR method was then used to test virus infections from field samples of sweet cherry in this paper. It will be quite helpful for plant quarantine and certification programs. To our knowledge, it is the first report of the multiplex RT-PCR assay for simultaneous detection of PNRSV, PDV, LChV-2 and CGRMV.

Elimination of mixed ‘Odontoglossum ringspot’ and ‘Cymbidium mosaic’ viruses from Phalaenopsis hybrid ‘V3’ through shoot-tip culture and protocorm-like body selection

Phalaenopsis is one of the most popular orchid plants in the global market. Several viruses have been reported to negatively impact its growth, yield and quality. A mixed infection of Odontoglossum ringspot virus and Cymbidium mosaic virus was detected in Phalaenopsis hybrid “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) plants in Taiwan. In the present communication, we report a relatively simple protocol for elimination of these two viruses using shoot-tip culture, and early isolation and selection of virus free protocorm-like body (PLB) through subcultures. The indexing of viruses in the field grown plants, PLBs and tissue culture plants was carried out by the Indirect enzyme-linked immunosorbent assay (ELISA), and one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). The induction of PLBs in shoot-tips was achieved on 1/2× Murashige and Skoog's basal medium supplemented with 1% sucrose and 0.9% Bacto-agar. PLBs could be maintained, proliferated and converted to plantlets on 1 g/L Hyponex medium supplemented with 1% sucrose, 6% potato pulp, 0.5 g/L tryptone, 0.25% activated charcoal and 0.6% Bacto-agar. The regenerated plantlets from virus-free PLBs acclimatized easily in a greenhouse and showed 100% survival rate. All the tissue culture-raised Phalaenopsis plants in the greenhouse tested negative for the two viruses. Our study demonstrates that some PLB lines selected at the first subculture as virus-free were found to be infected with virus at second subculture, however, re-occurrence of virus was never found in PLB lines at third subculture onwards. Hence, at least 3 subcultures are necessary to authenticate that the cultures are free of viruses. Simple culture media without plant growth regulators used in the present study minimizes the chances of somaclonal variations and ensures genetic uniformity of cultured plantlets, a highly desirable trait in the orchid industry. The method developed in the present study has potential of virus elimination, mass propagation of genetically uniform and virus-free Phalaenopsis plants.