scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

Detection of small round structured viruses in clinical and environmental samples by polymerase chain reaction

1995 ◽  
Vol 31 (5-6) ◽  
pp. 375-382 ◽  
Author(s):  
M. Wolfaardt ◽  
C. L. Moe ◽  
W. O. K. Grabow
Keyword(s):  
Polymerase Chain Reaction ◽  
Tap Water ◽  
Practical Method ◽  
Glass Wool ◽  
Polluted Water ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Elution Method

Norwalk (NV) and other small round structured viruses (SRSVs) have been identified as common causes of gastroenteritis. Outbreaks of Norwalk gastroenteritis have been associated with contaminated drinking water and food such as oysters and salads. The cloning and sequencing of the NV genome has made it possible to detect NV and related viruses by the reverse transcription-polymerase chain reaction (RT-PCR). We applied RT-PCR to detect SRSVs in faecal specimens from two gastroenteritis outbreaks in South Africa, designated “Christmas” and “Grootbrak” and were able to detect SRSVs in all of the three specimens from the Christmas outbreak and in two of 16 specimens from the Grootbrak outbreak. The RT-PCR procedure used appeared to be more sensitive for the detection of SRSVs in patient stool specimens than immune electron microscopy and NV antigen detection by enzyme linked immunosorbent assay. The RT-PCR procedure proved suitable for the detection of SRSVs in seeded samples of sewage, sewage sludge, river water, and tap water. However, sensitivity was lower for seeded samples of sewage and sludge than for tap water, which indicates interference by high levels of organic matter. The RT-PCR procedure was also used to show that small numbers of SRSVs can successfully be recovered from large volumes of water by means of a glass wool adsorption-elution method. Since no practical method is available for quantitation of the small numbers of SRSVs concerned, it was not possible to evaluate the efficiency of recovery. Although no SRSVs have been detected by direct testing of sewage and sludge samples, the results obtained in this study show that RT-PCR detection of SRSVs in sewage and polluted water environments is feasible, and that small numbers of the viruses can, like many other enteric viruses, successfully be recovered by means of a glass wool adsorption-elution method.

scholarly journals Molecular (real-time reverse transcription polymerase chain reaction) diagnosis of SARS-CoV-2 infections: complexity and challenges

2021 ◽  
Vol 45 (3) ◽  
pp. 135-142
Author(s):  
Shneh Sethi ◽  
Trinad Chakraborty
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Fatal Outcome ◽  
Respiratory Illness ◽  
World Health ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Pcr Assays

Abstract The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first recorded in Wuhan, China. The World Health Organization initially classified COVID-19 as a public health emergency and subsequently declared the disease a global pandemic. COVID-19 can take at least three distinct forms: severe acute distress syndrome with a potentially fatal outcome, mild respiratory illness (pneumonia with eventual recovery) and asymptomatic infection. All three disease forms have the potential to transmit the infection to healthy contacts. At present, real-time reverse transcription polymerase chain reaction (RT-PCR) is the only available laboratory tool to confirm the presence of viral RNA in patient specimens. These assays are designed to detect one or more (at least 2) SARS-CoV-2 RNA gene targets allowing the detection of the virus. Commercially available RT-PCR assays employ various gene targets of the viral genome in their assay systems. Additionally, there are differences in primer selection for the same gene region of SARS-CoV-2. At present, it is unclear whether the results from different RT-PCR assays are comparable in detecting the spectrum of COVID-19 manifestations. The purpose of the present article is twofold: first, to briefly focus on the findings of these reports; and second, to emphasize the various challenges and flaws that can potentially impact the diagnostic accuracy of RT-PCR testing for SARS-CoV-2.

scholarly journals Performance of Real-Time Reverse Transcription Polymerase Chain Reaction for the Detection of Foot-and-Mouth Disease Virus during Field Outbreaks in the United Kingdom in 2007

2009 ◽  
Vol 21 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Scott M. Reid ◽  
Katja Ebert ◽  
Katarzyna Bachanek-Bankowska ◽  
Carrie Batten ◽  
Anna Sanders ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
United Kingdom ◽  
Real Time ◽  
Reverse Transcription ◽  
Foot And Mouth Disease ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The United Kingdom ◽  
Polymerase Chain

Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). The present report describes the practical steps undertaken to deploy a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to process the samples received during the outbreaks of FMD in the United Kingdom in 2007. Two independent real-time RT-PCR assays targeting different regions (5′UTR and 3D) of the FMD virus (FMDV) genome were used to confirm the presence of FMDV in clinical samples collected from the first infected premises. Once the FMDV strain responsible had been sequenced, a single real-time RT-PCR assay (3D) was selected to test a total of 3,216 samples, including material from all 8 infected premises. Using a 96-well automated system to prepare nucleic acid template, up to 84 samples could be processed within 5 hr of submission, and up to 269 samples were tested per working day. A conservative cut-off was used to designate positive samples, giving rise to an assay specificity of 99.9% or 100% for negative control material or samples collected from negative premises, respectively. For the first time, real-time RT-PCR results were used to recognize preclinical FMD in a cattle herd. Furthermore, during the later stages of the outbreaks, the real-time RT-PCR assay supported an active surveillance program within high-risk cattle herds. To the authors' knowledge, this is the first documented use of real-time RT-PCR as a principal laboratory diagnostic tool following introduction of FMD into a country that was FMD-free (without vaccination) and highlights the advantages of this assay to support control decisions during disease outbreaks.

Interlaboratory Evaluation of a New Reverse Transcriptase Polymerase Chain Reaction–Based Enzyme-Linked Immunosorbent Assay for the Detection of Circulating Melanoma Cells: A Multicenter Study of the Dermatologic Cooperative Oncology Group

2001 ◽  
Vol 19 (6) ◽  
pp. 1723-1727 ◽  
Author(s):  
U. Reinhold ◽  
C. Berkin ◽  
A.-K. Bosserhoff ◽  
A. Deutschmann ◽  
C. Garbe ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Tumor Cells ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Blood Samples ◽  
Melanoma Patients ◽  
Polymerase Chain ◽  
Interlaboratory Reproducibility

PURPOSE: Reverse transcription-polymerase chain reaction (RT-PCR)–based detection of tyrosinase mRNA is the most frequently used laboratory method for the detection of circulating tumor cells in melanoma patients. However, previously published results showed considerable variability in the PCR positivity rates. MATERIALS AND METHODS: We designed a collaborative study to assess the sensitivity, specificity, and clinical relevance of a new standardized RT-PCR–based enzyme-linked immunosorbent assay (ELISA) for the detection of circulating melanoma cells. Blood samples of healthy donors mixed with cells of a melanoma cell line were prepared in a blinded fashion, and aliquots were sent to seven participating laboratories experienced in RT-PCR. RESULTS: The results demonstrate a high sensitivity (1 melanoma cell/mL blood) and specificity (no false-negatives and 7.4% [2 of 28] false-positives) of the assay and a satisfactory rate of interlaboratory reproducibility. The analysis of aliquots of blinded samples derived from 60 melanoma patients identified tyrosinase mRNA in 17 of 60 (28.3%): three (20%) of 15 stage I patients, two (13.3%) of 15 stage II patients, five (35.7%) of 14 stage III patients, and seven (43.8%) of 16 stage IV patients. The interlaboratory reproducibility of positive samples, however, was extremely low and indicates the presence of low amounts of target mRNA. CONCLUSION: Reverse transcriptase-PCR ELISA has a high sensitivity and specificity for the detection of tyrosinase mRNA in peripheral blood cells. The low interlaboratory reproducibility for the detection of tumor cells in blood samples of melanoma patients, however, raises the question of relevance of this assay for clinical use.

scholarly journals Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction

2006 ◽  
Vol 96 (3) ◽  
pp. 320-325 ◽  
Author(s):  
Nieves Capote ◽  
M. Teresa Gorris ◽  
M. Carmen Martínez ◽  
Margarita Asensio ◽  
Antonio Olmos ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Monoclonal Antibodies ◽  
Real Time ◽  
Reverse Transcription ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Japanese Plum ◽  
Polymerase Chain

The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3′ minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected.

scholarly journals Virus Nipah

2016 ◽  
Vol 8 (2) ◽  
Author(s):  
Novie H. Rampengan
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Immunosorbent Assay ◽  
Nipah Virus ◽  
Specific Treatment ◽  
The Philippines ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract: Nipah virus caused outbreaks in Malaysia and Singapore in 2009 with a high mortality rates. It also erupted in Bangladesh, India, and the Philippines. Nipah virus infection varies from asymptomatic to severe manifestation with a mortality rate varies from 38% to 80%. Diagnosis can be confirmed by using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemical examination, enzyme-linked immunosorbent assay (ELISA), and neutralization test. There is still neither vaccine nor specific treatment for the Nipah virus so farKeywords: Nipah virus, signs and symptoms, diagnosisAbstrak: Virus Nipah menimbulkan outbreak di Malaysia dan Singapura tahun 2009 dengan angka kematian yang tinggi. Selain itu virus Nipah juga menimbulkan outbreak di Bangladesh, India, dan Filipina. Infeksi virus Nipah dapat bervariasi dari asimtomatik sampai bermanifestasi klinis yang berat dengan angka kematian bervariasi dari 38%-80%. Diagnosis dapat ditegakkan dengan reverse transcriptase-polymerase chain reaction (RT-PCR), pemeriksaan imunohistokimia, enzyme-linked immunosorbent assay (ELISA), dan tes netralisasi. Sampai saat ini belum ada vaksin dan terapi spesifik untuk virus Nipah.Kata kunci: virus Nipah, gejala, diagnosis

Sign in / Sign up

Export Citation Format

Share Document