scholarly journals Unexpected cases in field diagnosis of African swine fever virus in Vietnam: The needs consideration when performing molecular diagnostic tests

2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Anh Duc Truong ◽  
Duc Viet Ly ◽  
Thi Hao Vu ◽  
Van Tuan Hoang ◽  
Thi Chinh Nguyen ◽  
...  
Keyword(s):  
Binding Site ◽  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
False Negative ◽  
African Swine Fever ◽  
Molecular Diagnostic ◽  
Conventional Pcr ◽  
Sequencing Method ◽  
Single Mismatch

Background: The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result.Aim: This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection.Methods: Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells.Results: The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study.Conclusion: A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences,  particularly for epidemiological surveillance of ASF. Keywords: African swine fever, PCR, Pigs, Real-time PCR, Vietnam

scholarly journals Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiyu Zhang ◽  
Ming Yao ◽  
Zhihui Tang ◽  
Daning Xu ◽  
Yan Luo ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Standard Deviation ◽  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
Simultaneous Detection ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Duck Tembusu Virus ◽  
Relative Standard

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

scholarly journals An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam

2020 ◽  
Vol 33 (10) ◽  
pp. 1683-1690
Author(s):  
Ha Thi Thanh Tran ◽  
Anh Kieu Dang ◽  
Duc Viet Ly ◽  
Hao Thi Vu ◽  
Tuan Van Hoang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Binding Sites ◽  
African Swine Fever Virus ◽  
False Negative ◽  
African Swine Fever ◽  
Clinical Samples ◽  
Probe Sequence ◽  
Field Samples ◽  
Polymerase Chain

Objective: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam.Methods: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE.Results: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78).Conclusion: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

scholarly journals A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study

2016 ◽  
Vol 55 (2) ◽  
pp. 180-184 ◽  
Author(s):  
Solène Le Gal ◽  
Florence Robert-Gangneux ◽  
Yann Pépino ◽  
Sorya Belaz ◽  
Céline Damiani ◽  
...  
Keyword(s):  
Real Time ◽  
Multicenter Study ◽  
Real Time Pcr ◽  
False Negative ◽  
False Negative Result ◽  
Pcr Assay

Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Food Control ◽  
2019 ◽  
Vol 98 ◽  
pp. 380-388 ◽  
Author(s):  
Xiaofu Wang ◽  
Ting Tang ◽  
Qingmei Miao ◽  
Shilong Xie ◽  
Xiaoyun Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Transgenic Rice ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Processed Foods ◽  
Conventional Pcr ◽  
Rice Line ◽  
Transgenic Rice Line

scholarly journals Building a molecular typing protocol for rs1801133 based on real-time PCR HRM technique

2019 ◽  
Vol 2 (3) ◽  
pp. 5-13
Author(s):  
Linh Thi Nhut Tran ◽  
My Thi Huynh Nguyen ◽  
Linh Nguy Hoang Le ◽  
Khoa Dang Le ◽  
Minh Hoang Nhat Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Neurological Diseases ◽  
Human Diseases ◽  
Chromosome 1 ◽  
Nucleotide Sequencing ◽  
Sequencing Method ◽  
Mgcl2 Concentration ◽  
Clear Differentiation ◽  
The Relationship

rs1801133 is a single nucleotide polymorphism (SNP) located in the sequence of MTHFR on human chromosome 1. The alleles of this SNP affect the activity of the MTHFR enzyme. People bearing C/T genotype have 66% activity of MTHFR while people with T/T genotype have only 25% activity. These reduced activities of MTHFR cause homocysteinemia. There are several publications on the relationship between homocysteinemia and human diseases such as cardiovascular disease, neurological diseases, abnormal fetus, infertility and cancer. In this study, we built a molecular protocol for genotyping rs1801133 using real-time PCR HRM technique. This protocol could be used for diagnosis of molecular mechanism of homocysteinemia causing the mentoned above diseases as well as for the study of the relationship between rs1801133 and other human diseases. We successfully designed the primer pairs for genotyping and nucleotide sequencing rs1801133 by real-time PCR HRM and Sanger sequencing method. We also examined the optimal MgCl2 concentration for clear differentiation of three rs1801133 genotypes. Performance characteristics of the real-time PCR HRM protocol included of specificity, repeatability, reproducibility was evaluated and it showed good results. Comparison of genotyping results of rs1801133 between the realtime PCR HRM method and the Sanger nucleotide sequencing method showed good concordances. Finally, this real-time PCR HRM protocol for rs1801133 genotyping was applied on 100 human DNA samples to evaluate the clinical utility of the protocol.

scholarly journals Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

2020 ◽  
Author(s):  
zhenhua Guo ◽  
Kunpeng Li ◽  
Songlin Qiao ◽  
Xinxin Chen ◽  
Ruiguang Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Limit Of Detection ◽  
African Swine Fever ◽  
Economic Losses ◽  
Clinical Samples ◽  
Diagnosis Method ◽  
Pcr Method ◽  
And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

scholarly journals False Negative Results in High Viremia Parvovirus B19-Samples Tested with Real-Time PCR

2010 ◽  
Vol 59 (2) ◽  
pp. 129-132 ◽  
Author(s):  
PIOTR GRABARCZYK ◽  
ALEKSANDRA KALIŃSKA ◽  
EWA SULKOWSKA ◽  
EWA BROJER
Keyword(s):  
Positive Result ◽  
Real Time ◽  
Real Time Pcr ◽  
Parvovirus B19 ◽  
False Negative ◽  
Immunocompromised Patients ◽  
Negative Results ◽  
False Negative Results ◽  
Parvovirus B 19 ◽  
The Way

Extremely high viremia is observed during some viruses infection, especialy in immunocompromised patients. False negative results of Parvovirus B 19 DNA tests performed with real-time PCR in high viremic samples are reported. The way of fluorescence diagrams analysis and algorithm of positive result confirmation to exclude such phenomenon are proposed.

scholarly journals Replication kinetics of Marek's disease vaccine virus in feathers and lymphoid tissues using PCR and virus isolation

2005 ◽  
Vol 86 (11) ◽  
pp. 2989-2998 ◽  
Author(s):  
Susan J. Baigent ◽  
Lorraine P. Smith ◽  
Richard J. W. Currie ◽  
Venugopal K. Nair
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
Marek's Disease ◽  
Pcr Analysis ◽  
Lymphoid Tissues ◽  
Avirulent Strain ◽  
Marek’S Disease ◽  
Virus Load ◽  
Kinetics Of

CVI988 (Rispens), an avirulent strain of Marek's disease virus, is the most widely used vaccine against Marek's disease. The kinetics of replication of CVI988 was examined in tissues of chickens vaccinated at either 1 day or 14 days of age and sampled regularly up to 28 days post-vaccination. Age at vaccination had no significant effect on the kinetics of CVI988 virus replication. During the cytolytic phase of infection (1–7 days), virus levels peaked in the spleen, bursa and thymus with very close correlation among these organs. Virus load in peripheral blood lagged behind and did not reach high levels. Significant numbers of virus genomes were detected in the feather tips only after 7 days, but subsequently rose to levels almost 103-fold greater than in the other tissues. This is the first accurate quantitative data for kinetics of CVI988 replication in a variety of tissues. There was good correlation between data from virus isolation and PCR, with real-time PCR being the preferred method for rapid, accurate and sensitive quantification of virus. Feathers were ideal for non-invasive sampling to detect and measure CVI988 in live chickens and, from 10 days onwards, virus load in feather tips was predictive of virus load in lymphoid tissues where immune responses will occur. The potential for real-time PCR analysis of feather samples for further investigation of the mechanism of vaccinal protection, and to assist optimization of vaccination regimes, is discussed.

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