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 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.

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

scholarly journals Comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue

2017 ◽  
Vol 9 (03) ◽  
pp. 177-181 ◽  
Author(s):  
Seema Gaikwad ◽  
Sandhya S. Sawant ◽  
Jayanthi S. Shastri
Keyword(s):  
Polymerase Chain Reaction ◽  
Nonstructural Protein ◽  
Antigen Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Nonstructural Protein 1 ◽  
Ns1 Antigen ◽  
Polymerase Chain ◽  
Sera Samples

Abstract INTRODUCTION: Early diagnosis of dengue is important for appropriate clinical management and vector control. Different serological tests based on the principle of immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used for detection of antigen and antibodies of dengue virus. The performance of these tests depends on the sensitivity and specificity. Hence, the study was undertaken to compare nonstructural protein-1 (NS1) antigen detection by rapid and ELISA with real-time polymerase chain reaction (RT-PCR) for diagnosis of dengue. MATERIALS AND METHODS: Prospective laboratory study was carried out on sera samples (n = 200) from clinically suspected cases of dengue. The sera samples were subjected for NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR. The results of rapid and ELISA tests were compared with real Time PCR. RESULTS: The sensitivity, specificity, positive, and negative predictive value of rapid dengue NS1 antigen test were 81.5%, 66.7%, 78.2%, and 71.1%, respectively whereas that of NS1 ELISA were 89.9%, 100%, 100%, and 94%, respectively. Concordance of Rapid NS1 and NS1 ELISA with PCR was 75.5% and 94%. DISCUSSION AND CONCLUSION: NS1 antigen ELISA can be implemented in diagnostic laboratories for diagnosis of dengue in the acute phase of illness. The test also has great potential value for use in epidemic situations, as it could facilitate the early screening of patients and limit disease expansion.

scholarly journals A Comparison Between Serological and Biological Assays in Detecting Grapevine Leafroll Associated Viruses

Plant Disease ◽  
1997 ◽  
Vol 81 (7) ◽  
pp. 799-801 ◽  
Author(s):  
Adib Rowhani ◽  
Jerry K. Uyemoto ◽  
Deborah A. Golino
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Multiple Infections ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vitis Vinifera L ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Biological Assays ◽  
Cabernet Franc ◽  
Polymerase Chain

The efficacy of the serological procedure enzyme-linked immunosorbent assay (ELISA) for detecting grapevine leafroll associated viruses (GLRaV types -1, -2, -3, and -4) was compared with indexing on Vitis vinifera L. cv. Cabernet Franc. Results of the biological assays confirmed the infectious nature of all grapevine sources testing positive by ELISA for GLRaV-1 (9 sources), GLRaV-2 (14 sources), and GLRaV-4 (14 sources), and the noninfectious nature of ELISA-negative grapevines (75 sources). However, among 57 sources testing positive by ELISA for GLRaV-3, or 24 sources with multiple infections, 8 and 1 sources, respectively, were negative by Cabernet Franc assays. Serological assays were repeated on all graft-inoculated indicators and only symptomatic ones reacted positively. Also, the 8 original GLRaV-3 sources that had tested positive by ELISA and negative by bioassay were found positive using immuno-capture/reverse transcription-polymerase chain reaction (IC/RT-PCR). The single multiple-infected source was not available for retesting. The distribution of GLRaV in infected grapevines was tested by assaying 20 to 40 samples per source of 36 plants infected with GLRaV-1, -2, -3, or -4. The incidence of GLRaV-positive canes as determined by ELISA ranged from 0 to 100%, suggesting that GLRaV can be unevenly distributed in chronically infected grapevines.

scholarly journals Sensitive Method for Testing Peanut Seed Lots for Peanut stripe and Peanut mottle viruses by Immunocapture-Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
2000 ◽  
Vol 84 (5) ◽  
pp. 559-561 ◽  
Author(s):  
A. G. Gillaspie ◽  
R. N. Pittman ◽  
D. L. Pinnow ◽  
B. G. Cassidy
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Capsid Region ◽  
Large Numbers ◽  
Peanut Stripe Virus ◽  
Pcr Method ◽  
Polymerase Chain

An immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) method was developed for testing peanut (Arachis hypogaea) seed lots for infection by Peanut stripe virus (PStV) and Peanut mottle virus (PeMV). A small slice was removed from each seed distal to the radicle of a random 100-seed sample, the slices were extracted in buffer and centrifuged, and a portion of the supernatant was incubated in a tube that had been coated with antiserum to either PStV or PeMV. Following immunocapture of the virus, the tube was washed, the RT-PCR mix (with primers designed from conserved sequences within the capsid region of each virus) was placed in the same tubes, and the test completed. Results obtained on 15 previously untested seed lots from the collection indicated good correlation between virus detected by the IC-RT-PCR method and virus detected from the same seed lots by enzyme-linked immunosorbent assay (ELISA). The IC-RT-PCR method detected three lots infected with PeMV and none with PStV from 106 seed lots grown in Ecuador (results confirmed by ELISA). The IC-RT-PCR method is more sensitive than ELISA (currently used on samples consisting of five seeds), is useful for testing large numbers of seed lots of peanut germ plasm, and could be adapted to test other plants and detect other viruses.

scholarly journals Development of a reverse transcription polymerase chain reaction for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals

2020 ◽  
Author(s):  
Eun-sil Park ◽  
Osamu Fujita ◽  
Masanobu Kimura ◽  
Akitoyo Hotta ◽  
Koichi Imaoka ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Neutralizing Antibodies ◽  
Clinical Symptoms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Severe Fever

AbstractBackgroundSevere fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble to those of SFTS patients and SFTS-contracted cats shows high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals.Methodology/Principle findingsFour primer sets were newly designed from consensus sequences constructed by 108 strains of SFTSV. A reverse transcription polymerase chain reaction (RT-PCR) with these four primer sets were successfully and specifically detected several clades of SFTSV. Their limits of detection are 1-10 copies/reaction. By this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients.Conclusion/SignificanceThis newly developed RT-PCR could detect SFTSV RNA of several clades from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.Author summaryThis study developed RT-PCR to detect SFTS animal cases. This assay could detect SFTSV RNA belonging to different clades. Cats diagnosed as SFTS had IgM or IgG, but not neutralizing antibodies. SFTS cat cases were distributed in the area where SFTS patients have been reported highly, indicating the establishment of the circulation of SFTSV in the environment. These diagnostic assays could be helpful tools to detect and not to miss SFTS animal cases.

scholarly journals Detection and Isolate Differentiation of Citrus tristeza virus in Infected Field Trees Based on Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
2004 ◽  
Vol 88 (6) ◽  
pp. 625-629 ◽  
Author(s):  
Zhipeng Huang ◽  
Phyllis A. Rundell ◽  
Xiong Guan ◽  
Charles A. Powell
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Sweet Orange ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus ◽  
Citrus Tristeza

Reverse transcription-polymerase chain reaction (RT-PCR) was compared with enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) for detection of non-decline-inducing and decline-inducing isolates of Citrus tristeza virus (CTV) in 21 field sweet orange and grapefruit plants on sour orange rootstock in Fort Pierce, FL. Among these samples, seven, six, and eight were infected with decline-inducing, non-decline-inducing, and both decline-inducing and non-decline-inducing isolates of CTV, respectively. However, there was not a good correlation between field symptoms and detection of the decline-inducing isolate. The results confirmed that RT-PCR is not only able to detect and differentiate decline-inducing and non-decline-inducing isolates of CTV in Florida, but also can detect both isolate types in a single field sweet orange or grapefruit tree. For most samples, results from RT-PCR, ELISA, and DTBIA were the same. However, the 320-bp fragments produced only from decline-inducing isolates were amplified from two sweet orange and two grapefruit samples that did not react with decline-inducing CTV-specific monoclonal antibody MCA13 in ELISA or DTBIA, indicating that RT-PCR has a higher sensitivity than these immunological tests for field sweet orange or grapefruit samples. Thus, RT-PCR is a simple, rapid, and specific procedure for CTV identification applicable to both research and diagnostic needs.

Sign in / Sign up

Export Citation Format

Share Document