scholarly journals A Novel Multiplex RT-PCR Assay for Simultaneous Detection of Dengue and Chikungunya Viruses

2020 ◽  
Vol 21 (21) ◽  
pp. 8281
Author(s):  
Mohammed Alimul Islam ◽  
Mohamed E. El Zowalaty ◽  
Sumaiya Islam ◽  
Mohiuddin Sharif ◽  
Md. Rajibur Rahman ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Cross Reactivity ◽  
Single Step ◽  
Epidemiological Surveillance ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Viral Genomes ◽  
Specificity And Sensitivity ◽  
Field Samples

The goal of the study was to develop a specific, sensitive, and cost-effective molecular RT-PCR diagnostic assay for the rapid and simultaneous detection of the serotypes of dengue virus (DENV) and Chikungunya virus (CHIKV) from sera of suspected febrile patients. A single-tube, single-step multiplex RT-PCR (mRT-PCR) assay was designed for the detection of viral genomes from clinical and field samples. Specificity and sensitivity of the mRT-PCR assay were evaluated against six different combinations using two reverse transcriptases (AMV-RT and RT-Ace) and three DNA polymerases (LA-Taq, rTaq, and Tth). Among the six combinations, the AMV-RT and LA-Taq combination was more specific and sensitive than other enzyme combinations for detecting viral genomes of DENV-1, DENV-2, DENV-3, and DENV-4 (p < 0.01), and for detecting viral genomes of CHIKV (p < 0.05). The detection limits of the mRT-PCR were 10 focus forming units (FFU) for CHIKV and 1 FFU, 20 FFU, 0.1 FFU, and 10 FFU for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. The primers used for the mRT-PCR did not show any cross-reactivity among the serotypes of DENV or CHIKV. Specificity and sensitivity of the newly developed mRT-PCR were validated using serum samples collected from febrile patients during dengue outbreaks in Bangladesh. The sensitivity for serotype detection of DENV and CHIKV was superior to the virus isolation method and the antigen detection method using the Dengue NS1-Ag assay. This novel mRT-PCR method can be used for molecular epidemiological surveillance of DENV and CHIKV in epidemic and endemic countries.

Detection of a novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome in Henan Province, China, using real-time reverse transcription PCR

2013 ◽  
Vol 62 (7) ◽  
pp. 1060-1064 ◽  
Author(s):  
Xueyong Huang ◽  
Licheng Liu ◽  
Yanhua Du ◽  
Hongxia Ma ◽  
Yujiao Mu ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Henan Province ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Reverse Transcription Pcr

A novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome (FTLS) was discovered in Henan Province, China. Here, we report the development of an assay for this novel bunyavirus based on real-time reverse transcription PCR (RT-PCR). The assay exhibited high sensitivity and specificity without cross-reactivity towards 13 other viruses that cause similar symptoms. To evaluate the performance of this assay in detecting clinical samples, we analysed 261 serum samples from patients in Henan Province between 2007 and 2010. Of these samples, 91.95 % were bunyavirus positive. Compared with serological assays, the real-time PCR assay was much more sensitive in identifying infected patients 1 to 7 days after the onset of symptoms.

scholarly journals Evaluation of a multiplex PCR method for the detection of porcine parvovirus types 1 through 7 using various field samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245699
Author(s):  
Seung-Chai Kim ◽  
Chang-Gi Jeong ◽  
Salik Nazki ◽  
Sim-In Lee ◽  
Ye-Chan Baek ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Epidemiological Surveillance ◽  
Serum Samples ◽  
Dna Viruses ◽  
Efficient Detection ◽  
Field Samples ◽  
Pcr Method ◽  
The 1960S ◽  
Mpcr Assay

Porcine parvoviruses (PPVs) are small, nonenveloped DNA viruses that are widespread in the global pig population. PPV type 1 (PPV1) is a major causative agent of reproductive failure and has been recognized since the 1960s. In recent decades, novel PPVs have been identified and designated as PPVs 2 through 7 (PPV2~PPV7). Although the epidemiological impacts of these newly recognized parvoviruses on pigs are largely unknown, continuous surveillance of these PPVs is needed. The aim of this study was to develop an improved and efficient detection tool for these PPVs and to assess the developed method with field samples. Using 7 sets of newly designed primers, a multiplex polymerase chain reaction (mPCR) protocol was developed for the simultaneous detection of the seven genotypes of PPV (PPV1~PPV7). The sensitivity of the mPCR assay was analyzed, and the detection limit was determined to be 3×103 viral copies. The assay was highly specific in detecting one or more of the viruses in various combinations in specimens. The mPCR method was evaluated with 80 serum samples, 40 lung or lymph node samples and 40 intestine or fecal samples. When applied to these samples, the mPCR method could detect the 7 viruses simultaneously, providing rapid results regarding infection and coinfection status. In conclusion, the developed mPCR assay can be utilized as an effective and accurate diagnostic tool for rapid differential detection and epidemiological surveillance of various PPVs in numerous types of field samples.

scholarly journals A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1358
Author(s):  
Brigitte Sigrist ◽  
Jessica Geers ◽  
Sarah Albini ◽  
Dennis Rubbenstroth ◽  
Nina Wolfrum
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Epidemiological Studies ◽  
Virus Species ◽  
Rt Pcr ◽  
P Gene ◽  
Field Samples ◽  
Causative Agents ◽  
Species Specific ◽  
Proventricular Dilatation Disease

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

2013 ◽  
Vol 189 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Yan ◽  
Heng-hui Wang ◽  
Lei Gao ◽  
Ji-mei Ji ◽  
Zhi-jie Ge ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Norovirus ◽  
One Step

scholarly journals Automated Low Cost Method of Quantification of Hepatitis C Virus

2015 ◽  
Vol 14 (3) ◽  
pp. 247-253
Author(s):  
Faiz MMT Marikar ◽  
Dammika Senevirathna ◽  
Neil Fernandopulle
Keyword(s):  
Viral Load ◽  
Hepatitis C ◽  
Blood Donors ◽  
Low Cost ◽  
Medical Science ◽  
Simultaneous Detection ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Viral Genomes ◽  
Post Therapy

This paper describes the development of a Dig-dUTP based multiplex real time RT-PCR for the simultaneous detection of HCV viral amount in plasma samples. Viral genomes were identified in the same sample by Dig-dUTP PCR 216 bp region. Analysis of known scalar concentrations of reference plasma indicated that the multiplex procedure detects at least 500 copies/ml of HCV. In addition, we also assayed HCV viral load in eighty co-infected patients and in fifteen blood donors, confirming the sensitivity and specificity of the assay. This method may represent a useful alternative method for the detection of HCV co-infection, reliable for a rapid and relatively inexpensive screening of blood donors. The assay may be used to determine post-therapy viral clearance.Bangladesh Journal of Medical Science Vol.14(3) 2015 p.247-253

scholarly journals A multiplex RT-PCR assay for rapid and simultaneous detection of four RNA viruses in swine

2019 ◽  
Vol 269 ◽  
pp. 38-42 ◽  
Author(s):  
Yan Zhao ◽  
Feifei Liu ◽  
Qingmei Li ◽  
Mengfan Wu ◽  
Lei Lei ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay

scholarly journals EVALUATION OF ELEVEN IMMUNOCHROMATOGRAPHIC ASSAYS FOR SARS-CoV-2 DETECTION: INVESTIGATING DENGUE CROSS-REACTION

2020 ◽  
Author(s):  
Beatriz Araujo Oliveira ◽  
Lea Campos de Oliveira ◽  
Franciane Mendes de Oliveira ◽  
Geovana Maria Pereira ◽  
Regina Maia de Souza ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Diagnostic Techniques ◽  
Cross Reactivity ◽  
Cross Reaction ◽  
List Type ◽  
Rt Pcr ◽  
Serum Samples ◽  
Igm Antibodies ◽  
Good Agreement

AbstractBackgroundCOVID-19 disease (Coronavirus disease 2019) caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is widespread worldwide, affecting more than 11 million people globally (July 6th, 2020). Diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and low cost alternative for monitoring the spread of COVID-19 in the population.MethodsHere we evaluate the sensitivity and specificity of eleven different immunochromatographic tests in 98 serum samples from confirmed cases of COVID-19 through RT-PCR and 100 negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using 20 serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests.ResultsOur results demonstrated agreement between immunochromatographic assays and RT-PCR, especially after 10 days since the onset of symptoms. The evaluation of IgG and IgM antibodies combined demonstrated a strong level of agreement (0.85) of IC assays and RT-PCR. It was observed cross-reactivity between Dengue and COVID-19 using four different IC assays for COVID-19 diagnosis. The specificity of IC assays to detected COVID-19 IgM antibodies using Dengue serum samples varied from 80% to 85%; the specificity of IgG detection was 100% and total antibody was 95%.ConclusionsWe found high sensitivity, specificity and good agreement of IC assays, especially after 10 days onset of symptoms. However, we detected cross-reactivity between Dengue and COVID-19 mainly with IgM antibodies demonstrating the need for better studies about diagnostic techniques for these diseases.HighlightsImmunochromatographic assays demonstrated high sensitivity and specificity and good agreement with the gold-standard RT-PCR;Increase in sensitivity and specificity of assays using samples collected after the 10th day of symptoms;Cross-reaction with Dengue serology in evaluation of IgM.

scholarly journals Simultaneous Detection of Drug-Induced Liver Injury Protein and microRNA Biomarkers Using Dynamic Chemical Labelling on a Luminex MAGPIX System

Analytica ◽  
2021 ◽  
Vol 2 (4) ◽  
pp. 130-139
Author(s):  
Antonio Marín-Romero ◽  
Mavys Tabraue-Chávez ◽  
Bárbara López-Longarela ◽  
Mario A. Fara ◽  
Rosario M. Sánchez-Martín ◽  
...  
Keyword(s):  
Liver Injury ◽  
Simultaneous Detection ◽  
Single Step ◽  
Qualitative And Quantitative Analysis ◽  
Serum Samples ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Qualitative And Quantitative ◽  
Post Marketing ◽  
Mechanistic Basis

Drug-induced liver injury (DILI) is a potentially fatal adverse event and a leading cause for pre- and post-marketing drug withdrawal. Several multinational DILI initiatives have now recommended a panel of protein and microRNA (miRNA) biomarkers that can detect early liver injury and inform about mechanistic basis. This manuscript describes the development of seqCOMBO, a unique combo-multiplexed assay which combines the dynamic chemical labelling approach and an antibody-dependant method on the Luminex MAGPIX system. SeqCOMBO enables a versatile multiplexing platform to perform qualitative and quantitative analysis of proteins and miRNAs in patient serum samples simultaneously. To the best of our knowledge, this is the first method to profile protein and miRNA biomarkers to diagnose DILI in a single-step assay.

scholarly journals Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1975
Author(s):  
Petra Drzewnioková ◽  
Francesca Festa ◽  
Valentina Panzarin ◽  
Davide Lelli ◽  
Ana Moreno ◽  
...  
Keyword(s):  
Literature Review ◽  
In Silico ◽  
Host Shift ◽  
Molecular Tools ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Field Samples ◽  
Low Sensitivity

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and β showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and β-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10−5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, β-, and γ-.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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