Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

1995 ◽  
Vol 58 (12) ◽  
pp. 1357-1362 ◽  
Author(s):  
LEE-ANN JAYKUS ◽  
RICARDO DE LEON ◽  
MARK D. SOBSEY
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Hepatitis A Virus ◽  
Enteric Virus ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Ammonium Bromide ◽  
Rt Pcr ◽  
Initial Inoculum ◽  
Ctab Precipitation

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

Detection Methods for Human Enteric Viruses in Representative Foods†

2000 ◽  
Vol 63 (12) ◽  
pp. 1738-1744 ◽  
Author(s):  
PARIS R. LEGGITT ◽  
LEE-ANN JAYKUS
Keyword(s):  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Food Sample ◽  
Viral Rna ◽  
Detection Methods ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Viral Contamination ◽  
Initial Inoculum ◽  
Human Enteric Viruses

Although viral foodborne disease is a significant problem, foods are rarely tested for viral contamination, and when done, testing is limited to shellfish commodities. In this work, we report a method to extract and detect human enteric viruses from alternative food commodities using an elution-concentration approach followed by detection using reverse transcription-polymerase chain reaction (RT-PCR). Fifty-gram lettuce or hamburger samples were artificially inoculated with poliovirus type 1 (PV1), hepatitis A virus (HAV), or the Norwalk virus and processed by the sequential steps of homogenization, filtration, Freon extraction (hamburger), and polyethylene glycol (PEG) precipitation. To reduce volumes further and remove RT-PCR inhibitors, a secondary PEG precipitation was necessary, resulting in an overall 10- to 20-fold sample size reduction from 50 g to 3 to 5 ml. Virus recoveries in secondary PEG concentrates ranged from 10 to 70% for PV1 and 2 to 4% for HAV as evaluated by mammalian cell culture infectivity assay. Total RNA from PEG concentrates was extracted to a small volume (30 to 40 μl) and subjected to RT-PCR amplification of viral RNA sequences. Detection limit studies indicated that viral RNA was consistently detected by RT-PCR at initial inoculum levels ≥102 PFU/50-g food sample for PV1 and ≥103 PFU/50-g food sample for HAV. In similar studies with the Norwalk virus, detection at inoculum levels ≥1.5 × 103 PCR-amplifiable units/50-g sample for both food products was possible. All RT-PCR amplicons were confirmed by subsequent Southern hybridization. The procedure reported represents progress toward the development of methods to detect human enteric viral contamination in foods other than shellfish.

Virion Concentration Method for the Detection of Human Enteric Viruses in Extracts of Hard-Shelled Clams†

1998 ◽  
Vol 61 (4) ◽  
pp. 458-465 ◽  
Author(s):  
ALISSA B. DIX ◽  
LEE-ANN JAYKUS
Keyword(s):  
Cell Culture ◽  
Hepatitis A Virus ◽  
Direct Detection ◽  
Enteric Viruses ◽  
Precipitation Method ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Genomic Amplification ◽  
Initial Inoculum ◽  
Human Enteric Viruses

A method to extract and concentrate intact human enteric viruses from oyster extracts for detection using reverse transcription-polymerase chain reaction (RT-PCR) was applied to hard-shelled clams (Mercenaria mercenaria). Fifty-gram clam samples were processed by an adsorption-elution-precipitation method and then seeded with 101 to 105 PFU of poliovirus 1 (PV1) and/or hepatitis A virus (HAV). Seeded viruses in extracts were purified by fluorocarbon (Freon) extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Efficiency of virion recovery from PEG precipitates was dependent upon PEG concentration and elution buffer volume, with optimized variables yielding recoveries as high as 99% for PV1 and 45% for FIAV, as evaluated by cell culture infectivity assay. To further concentrate viruses, remove inhibitors, and reduce sample volumes, the protein-precipitating agent Pro-Cipitate was used in an adsorption-elution-precipitation scheme. The final concentrate was of low volume (<1 ml) and directly compatible with viral genomic amplification using RT-PCR. When extracts from 50-g clam samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 103 PFU for PV1 and HAV. Corresponding virus recoveries based on cell culture infectivity were 7 to 50% and 0.3 to 8% for PV1 and HAV, respectively. When extracts of clams were artificially contaminated with the Norwalk virus, direct detection of virion RNA using RT-PCR and subsequent oligoprobe hybridization was possible at levels as low as 450 RT-PCR amplifiable units of the Norwalk virus per extract of 50-g clam sample.

scholarly journals Combined SARS-CoV-2 nucleic acid amplification testing and respiratory virus panel RT-PCR on the Hologic Panther Fusion system

2021 ◽  
Vol 138 ◽  
pp. 104792
Author(s):  
Bryan A. Stevens ◽  
Catherine A. Hogan ◽  
Kenji O. Mfuh ◽  
Ghazala Khan ◽  
Malaya K. Sahoo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Respiratory Virus ◽  
Nucleic Acid Amplification ◽  
Fusion System ◽  
Rt Pcr ◽  
Nucleic Acid Amplification Testing

scholarly journals Molecular Diagnosis in Differentiating Active and Inactive Forms of Hepatitis B Virus Carriers

2018 ◽  
Vol 26 (10) ◽  
pp. 41-45
Author(s):  
Salah Tofik Jalal Balaky ◽  
Saeed Ghulam Hussain ◽  
Amer Ali Khaleel ◽  
Furat Tahseen Sabeer ◽  
Ahang Hasan Mawlood
Keyword(s):  
Nucleic Acid ◽  
Hepatitis B ◽  
Cross Sectional Study ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Detection Methods ◽  
Rt Pcr ◽  
Cross Sectional ◽  
Blood Samples ◽  
Acid Test

Background & objectives: Introducing a nucleic acid test program is aimed to diagnose and reduces the risk of viral infection or transmission. DNA assay for HBV can detect infection in the windows period, chronic occult infection and can discriminate between active and inactive HBV infection. This cross-sectional study designed to diagnose, analyze HBV infection and to differentiate active from inactive infection based on viral DNA detection. Methods: Blood samples were collected from 256 patients previously diagnosed on the clinical ground as hepatitis B seropositive in Erbil Central Lab. The viral nucleic acid quantitative assessment was done for the collected samples using RT-PCR. Q-square was performed for statistical analysis. Results: Out of 256 collected blood samples 93 (36.3%) showed HBV-DNA positive titers above 50 IU/ml. Among positive subjects, 67 (72.04%) was categorized as inactive carriers (˂ 2000-20.000 IU/ml HBV-DNA titers). Conclusions: The data produced from this study confirmed the importance of the RT-PCR technique in sensitivity and reliability as a superior diagnostics tool specifically in differentiating active from inactive HBV carriers.

scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

Role of Covid Antigen in the Diagnosis of Covid Positive in Obstetric Patients

2021 ◽  
Vol 15 (10) ◽  
pp. 3356-3358
Author(s):  
Ambreen Fatima ◽  
Nidda Yaseen ◽  
Amna Fareed ◽  
Kashif Ali Samin ◽  
Shumaela Kanwal ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Early Detection ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
False Negative ◽  
Nucleic Acid Amplification ◽  
Antigen Test ◽  
Rt Pcr ◽  
Cross Sectional ◽  
Specificity And Sensitivity

Background and Aim: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid emergence postured significant challenges on the health system in recent years. The early detection of cases is thought to be critical in preventing this pandemic by coronavirus disease (COVID-19), especially important in the obstetrical population due to theirs numerous interactions with another parturient when hospitalized for delivery. Therefore, the present study aimed to assess the COVID antigen test performance in COVID-positive obstetrics patients. Materials and Methods: This cross-sectional study was conducted on 1296 Covid-19 asymptomatic women admitted to the Obstetrics and Gynaecology Department of Muhammad Teaching Hospital & Medical College, Peshawar and Fauji Foundation Hospital, Rawalpindi for the duration of six months from February 2021 to July 2021. Antigen-based test rapid diagnostic test (RDT) was used for screening out COVID-19 positive obstetrics patients or women through nasopharyngeal swabs. Women with negative rapid antigen test results were confirmed with RT-polymers chain reaction test of nucleic acid amplification tests (NAAT). Ethical approval and informed consent were taken from the hospital ethical committee and each individual respectively. All the known positive COVID-19 patients during admission were excluded. SPSS version 24 was used for data analysis. Results: The overall prevalence of rapid antigen-positive tested patients was 13.2% (171/1296). The prevalence of positive tested women through rapid antigen test, Nucleic Acid Amplification Test (NAAT), and RT-PCR were 27 (2.1%), 51 (3.9%), and 93 (7.2%) respectively. Of the total 1296 rapid antigen tests, 27 were positive, and the false-negative confirmed positive by NAAT was 144.Thus the sensitivity of the rapid antigen test was 15.8% and the negative predictive value was 93.7%. Of the total 298 Nucleic Acid Amplification Tested had sensitivity and negative predictive value of 89.6% and 99.06% respectively. RT-PCR was carried out on 972 patients, positive diagnosed cases were 36 while 15 were initially negative and were positive with the test was repeated. The sensitivity and negative predictive value was 71.45% and 95.8% respectively. Conclusion: Our study found that Ag-RDT plays a significant role in SARS-CoV-2 early detection in infected individuals, with high specificity and sensitivity to disease infectious stage, whether symptomatic or asymptomatic, and can be used as a decision supported tool. Early detection of COVID-19 status in women admitted for delivery could benefit neonatal protection care. Keywords: Covid-19; Rapid antigen test; RT-PCR test

Transition from Antigenemia to Quantitative Nucleic Acid Amplification Testing in Kidney Transplant Recipients Receiving Preemptive Therapy for Cytomegalovirus Infection.

2021 ◽  
Author(s):  
Mônica Rika Nakamura ◽  
Lúcio R. Requião-Moura ◽  
Roberto Mayer Gallo ◽  
Camila Botelho ◽  
Júlia Taddeo ◽  
...  
Keyword(s):  
Glomerular Filtration Rate ◽  
Nucleic Acid ◽  
Acute Rejection ◽  
Glomerular Filtration ◽  
Filtration Rate ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Cmv Infection ◽  
Preemptive Treatment ◽  
The Impact

Abstract Due to the high costs, the strategy to reduce the impact of cytomegalovirus (CMV) after kidney transplant (KT) involves preemptive treatment in low and middle-income countries. Thus, this retrospective cohort study compared the performance of antigenemia transitioned to quantitative nucleic acid amplification testing, RT-PCR, in KT recipients receiving preemptive treatment as a strategy to prevent CMV infection. Between 2016 and 2018, 363 patients were enrolled and received preemptive treatment based on antigenemia (n=177) or RT-PCR (n=186). The primary outcome was CMV infection or disease. There were no differences in one-year cumulative incidence of CMV-related events (50.8% vs. 44.1%, P=0.20), neither in time to diagnosis (47.0 vs. 47.0 days) among patients conducted by antigenemia vs. RT-PCR, respectively. The length of CMV first treatment was longer with RT-PCR (20.0 vs. 27.5 days, P<0.001), while the rate of retreatment was not different (14.7% vs. 11.8%, P=0.48). In the Cox regression, the variables associated with CMV-related events were acute rejection within 30 days (HR=2.05, p=0.01) and 30-day glomerular filtration rate (HR=0.98, p<0.001). In conclusion, acute rejection and glomerular filtration rate are risk factors for CMV infection and disease, showing comparable performance in the impact of CMV-related events between antigenemia and RT-PCR for preemptive treatment.

scholarly journals Keeping up with new technology: new approaches to diagnosis of Chlamydia infection

1996 ◽  
Vol 42 (5) ◽  
pp. 809-812 ◽  
Author(s):  
W D LeBar
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Clinical Utility ◽  
New Technology ◽  
Nucleic Acid Amplification ◽  
Chlamydia Infection ◽  
Chain Reaction ◽  
Sexually Transmitted ◽  
The Us ◽  
Infected Sample

Abstract Chlamydia trachomatis infections are among the most common sexually transmitted infections in the US today. One of the keys to the prevention of C. trachomatis infection rests on the ability to make this diagnosis on the basis of accurate laboratory testing. For many years the standard for diagnosis of C. trachomatis infections has been isolation in tissue culture. Numerous nonculture methods, including enzyme immunoassay, have been used as an alternative to cell culture. The performance characteristics of these tests have all been compared with a standard, cell culture, which at best will detect 90% of positive specimens. Nucleic acid amplification techniques, including PCR and ligase chain reaction, have been recently introduced. The advantage of these tests is their ability to detect 10-20% more positive specimens when compared with culture or confirmed nonculture methods performed with a single specimen. The sensitivity of amplified tests also allows us to test specimens from multiple sites (endocervix, urethra, urine), which expands our standard from an infected sample to detection of an infected patient. Tests based on amplified nucleic acid technology have greatly improved our ability to diagnose urogenital C. trachomatis infection. The use of an expanded standard will help us accurately define the true performance and clinical utility of nonculture Chlamydia diagnostic tests.

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