scholarly journals SARS-CoV-2 Diagnostic Tests: Algorithm and Field Evaluation From the Near Patient Testing to the Automated Diagnostic Platform

2021 ◽  
Vol 8 ◽  
Author(s):  
Nicolas Yin ◽  
Cyril Debuysschere ◽  
Marc Decroly ◽  
Fatima-Zohra Bouazza ◽  
Vincent Collot ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Reverse Transcription ◽  
Diagnostic Tests ◽  
Point Of Care ◽  
Diagnostic Algorithm ◽  
Molecular Techniques ◽  
Diagnostic Tools ◽  
Target Populations ◽  
Reverse Transcription Pcr ◽  
Rapid Tests

Introduction: Since the first wave of COVID-19 in Europe, new diagnostic tools using antigen detection and rapid molecular techniques have been developed. Our objective was to elaborate a diagnostic algorithm combining antigen rapid diagnostic tests, automated antigen dosing and rapid molecular tests and to assess its performance under routine conditions.Methods: An analytical performance evaluation of four antigen rapid tests, one automated antigen dosing and one molecular point-of-care test was performed on samples sent to our laboratory for a SARS-CoV-2 reverse transcription PCR. We then established a diagnostic algorithm by approaching median viral loads in target populations and evaluated the limit of detection of each test using the PCR cycle threshold values. A field performance evaluation including a clinical validation and a user-friendliness assessment was then conducted on the antigen rapid tests in point-of-care settings (general practitioners and emergency rooms) for outpatients who were symptomatic for <7 days. Automated antigen dosing was trialed for the screening of asymptomatic inpatients.Results: Our diagnostic algorithm proposed to test recently symptomatic patients using rapid antigen tests, asymptomatic patients using automated tests, and patients requiring immediate admission using molecular point-of-care tests. Accordingly, the conventional reverse transcription PCR was kept as a second line tool. In this setting, antigen rapid tests yielded an overall sensitivity of 83.3% (not significantly different between the four assays) while the use of automated antigen dosing would have spared 93.5% of asymptomatic inpatient screening PCRs.Conclusion: Using tests not considered the “gold standard” for COVID-19 diagnosis on well-defined target populations allowed for the optimization of their intrinsic performances, widening the scale of our testing arsenal while sparing molecular resources for more seriously ill patients.

scholarly journals Identification of Encephalomyocarditis Virus in Clinical Samples by Reverse Transcription-PCR Followed by Genetic Typing Using Sequence Analysis

1998 ◽  
Vol 36 (12) ◽  
pp. 3463-3467 ◽  
Author(s):  
H. Vanderhallen ◽  
F. Koenen
Keyword(s):  
Sequence Analysis ◽  
Reverse Transcription ◽  
Molecular Techniques ◽  
Encephalomyocarditis Virus ◽  
Clinical Samples ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Field Samples ◽  
Pcr Targeting

The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3′ end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.

scholarly journals Diagnostic accuracy of two commercial SARS-CoV-2 Antigen-detecting rapid tests at the point of care in community-based testing centers

2020 ◽  
Author(s):  
Alice Berger ◽  
Marie Therese Ngo Nsoga ◽  
Francisco Javier Perez-Rodriguez ◽  
Yasmine Abi Aad ◽  
Pascale Sattonnet-Roche ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Point Of Care ◽  
Rapid Test ◽  
Diagnostic Value ◽  
Rapid Diagnostic Tests ◽  
Ease Of Use ◽  
Rt Pcr ◽  
Test Device ◽  
Community Based ◽  
Rapid Tests

AbstractBackgroundAntigen-detecting rapid diagnostic tests for SARS-CoV-2 offer new opportunities for the quick and laboratory-independent identification of infected individuals for control of the SARS-CoV-2 pandemic.MethodsWe performed a prospective, single-center, point of care validation of two antigen-detecting rapid diagnostic tests (Ag-RDT) in comparison to RT-PCR on nasopharyngeal swabs.FindingsBetween October 9th and 23rd, 2020, 1064 participants were enrolled. The Panbio™Covid-19 Ag Rapid Test device (Abbott) was validated in 535 participants, with 106 positive Ag-RDT results out of 124 positive RT-PCR individuals, yielding a sensitivity of 85.5% (95% CI: 78.0–91.2). Specificity was 100.0% (95% CI: 99.1–100) in 411 RT-PCR negative individuals. The Standard Q Ag-RDT (SD Biosensor, Roche) was validated in 529 participants, with 170 positive Ag-RDT results out of 191 positive RT-PCR individuals, yielding a sensitivity of 89.0% (95%CI: 83.7–93.1). One false positive result was obtained in 338 RT-PCR negative individuals, yielding a specificity of 99.7% (95%CI: 98.4–100). For individuals presenting with fever 1-5 days post symptom onset, combined Ag-RDT sensitivity was above 95%.InterpretationWe provide an independent validation of two widely available commercial Ag-RDTs, both meeting WHO criteria of ≥80% sensitivity and ≥97% specificity. Although less sensitive than RT-PCR, these assays could be beneficial due to their rapid results, ease of use, and independence from existing laboratory structures. Testing criteria focusing on patients with typical symptoms in their early symptomatic period onset could further increase diagnostic value.FundingFoundation of Innovative Diagnostics (FIND), Fondation privée des HUG, Pictet Charitable Foundation.

scholarly journals New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
William S. Probert ◽  
Jill K. Hacker
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Outbreak Detection ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Laboratory Surveillance

ABSTRACTLaboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

scholarly journals Generation of scFv-Monoclonal Antibody Avian Influenza Diagnostic tests

2019 ◽  
Vol 24 (1) ◽  
Author(s):  
Simson Tarigan ◽  
. Sumarningsih
Keyword(s):  
Monoclonal Antibody ◽  
Avian Influenza ◽  
Polyclonal Antibody ◽  
Diagnostic Tests ◽  
Influenza A ◽  
Point Of Care ◽  
Influenza Viruses ◽  
Diagnostic Tools ◽  
Phage Display Technology ◽  
E Coli

<p><span lang="EN-US">The need for rapid diagnostic tools or point- of- care diagnostic tests for Avian Influenza in Indonesia is very high and the price of these imported diagnostic tools is very expensive. As a result, a large budget requires to provide the needs. The main component of a rapid diagnostic tool is the monoclonal antibody (mAb) specifically recognized influenza viruses. The objective of this study was to produce mAb that can recognize all subtypes of Avian Influenza viruses using the phage display technology. Influenza-A focused scFv commercial library was panned using alternating recombinant H1N1 NP and H5N1 virions. Whereas, bacteriophages bound to the panning baits were eluted with serum from H5N1-infected chickens. Phagemid from suppressor E. coli (TG1) infected with bacteriophage displaying anti-NP on its surface was isolated and then transformed into a non-suppressor E. coli (HB2151) to express NP-scFv. Monoclonal NP-scFv antibody with a molecular weight of about 27 kDa was purified from the culture supernatant using a nickel-chromatography column. The amount of pure NP-scFv obtained was around 1.2 mg /L culture. As an additional component for its use in immunoassays, antibody to NP-scFv was produced in rabbits. The generating polyclonal antibody recognized the NP-scFv specifically and sensitively. The anti-NP-scFv monoclonal antibody and the anti rabbit scFv polyclonal antibody produced in this study are envisaged appropriate for the development of diagnostic tools for point-of-care for Avian Influenza.</span></p>

scholarly journals Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dilip Kumar Ghosh ◽  
Sunil B. Kokane ◽  
Siddarame Gowda
Keyword(s):  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Point Of Care ◽  
Lateral Flow ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Immunochromatographic Assay ◽  
Tristeza Virus ◽  
Lateral Flow Immunochromatographic Assay ◽  
Citrus Tristeza

AbstractTristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15–20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

scholarly journals Mammaglobin as a Novel Breast Cancer Biomarker: Multigene Reverse Transcription-PCR Assay and Sandwich ELISA

2004 ◽  
Vol 50 (11) ◽  
pp. 2069-2076 ◽  
Author(s):  
Barbara K Zehentner ◽  
David H Persing ◽  
Amadou Deme ◽  
Papa Toure ◽  
Stephen E Hawes ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Reverse Transcription ◽  
Sandwich Elisa ◽  
Clinical Staging ◽  
Diagnostic Tools ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Blood Samples ◽  
Mixed Control ◽  
Reverse Transcription Pcr

Abstract Background: The aim of this study was to examine the potential usefulness of a mammaglobin multigene reverse transcription-PCR (RT-PCR) assay and a mammaglobin sandwich ELISA as diagnostic tools in breast cancer. Methods: We studied peripheral blood samples from 147 untreated Senegalese women with biopsy-confirmed breast cancer and gathered patient information regarding demographic, and clinical staging of disease. The samples were tested for mammaglobin and three breast cancer-associated gene transcripts by a multigene real-time RT-PCR assay and for serum mammaglobin protein by a sandwich ELISA assay. Results: In 77% of the breast cancer blood samples, a positive signal was obtained in the multigene RT-PCR assay detecting mammaglobin and three complementary transcribed genes. Fifty samples from healthy female donors tested negative. Significant correlations were found between mammaglobin protein in serum, presence of mammaglobin mRNA-expressing cells in blood, stage of disease, and tumor size. Circulating mammaglobin protein was detected in 68% of the breast cancer sera, and was increased in 38% in comparison with a mixed control population. The RT-PCR assay and the ELISA for mammaglobin produced a combined sensitivity of 84% and specificity of 97%. Conclusion: The ELISA and RT-PCR for mammaglobin and mammaglobin-producing cells could be valuable tools for diagnosis and prognosis of breast cancer.

scholarly journals Performance Evaluation of the CareGENETM Zika Virus Reverse Transcription-PCR Kit for Urine Specimen

2021 ◽  
Vol 11 (4) ◽  
pp. 267-274
Author(s):  
Gun Woo Kim ◽  
Borae G. Park ◽  
Seung Yeon Kwak ◽  
Woong Sik Jang ◽  
Jeounghun Nam ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Urine Specimen ◽  
Reverse Transcription Pcr

scholarly journals PCR past, present and future

BioTechniques ◽  
2020 ◽  
Vol 69 (4) ◽  
pp. 317-325 ◽  
Author(s):  
Hanliang Zhu ◽  
Haoqing Zhang ◽  
Ying Xu ◽  
Soňa Laššáková ◽  
Marie Korabečná ◽  
...  
Keyword(s):  
Forensic Science ◽  
Real Time ◽  
High Throughput ◽  
Reverse Transcription ◽  
Gold Standard ◽  
Point Of Care ◽  
Reverse Transcription Pcr ◽  
History Of ◽  
Pcr Method

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.

New Approaches for Detection of Mycoplasma iowae Infection in Turkeys

1995 ◽  
Author(s):  
Sharon Levisohn ◽  
Mark Jackwood ◽  
Stanley Kleven
Keyword(s):  
Experimental Infection ◽  
Mixed Infection ◽  
Diagnostic Tests ◽  
Phenotypic Variability ◽  
Molecular Techniques ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Mycoplasma Iowae ◽  
Pcr Test

Mycoplasma iowae (Mi) is a pathogenic avian mycoplasma which causes mortality in turkey embryos and as such has clinical and economic significance for the turkey breeder industry. Control of Mi infection is severely hampered by lack of adequate diagnostic tests, together with resistance to most antibiotics and resilience to environment. A markedly high degree of intra-species antigenic variation also contributes to difficulties in detection and control of infection. In this project we have designed an innovative gene-based diagnostic test based on specific amplification of the 16S rRNA gene of Mi. This reaction, designed Multi-species PCR-RFLP test, also amplifies the DNA of the pathogenic avian mycoplasmas M. gallisepticum (Mg) and M. synoviae (Ms). This test detects DNA equivalent to about 300 cfu Mi or either of the other two target mycoplasmas, individually or in mixed infection. It is a quick test, applicable to a wide variety of clinical samples, such as allantoic fluid or tracheal or cloacal swab suspensions. Differential diagnosis is carried out by gel electro-phoresis of the PCR amplicon digested with selected restriction enzymes (Restriction Fragment Length Polymorphism). This can also be readily accomplished by using a simple Dot-Blot hybridization assay with digoxigenin-labeled oligonucleotide probes reacting specifically with unique Mi, Mg or Ms sequences in the PCR amplicon. The PCR/OLIGO test increased sensitivity by at least 10-fold with a capacity for rapid testing of large numbers of samples. Experimental infection trials were carried out to evaluate the diagnostic tools and to study pathogenesis of Mi infection. Field studies and experimental infection of embryonated eggs indicated both synergistic and competitive interaction of mycoplasma pathogens in mixed infection. The value of the PCR diagnostic tests for following the time course of egg transmission was shown. A workable serological test (Dot Immunobinding Assay) was also developed but there was no clear-cut evidence that infected turkeys develop an immune response. Typing of a wide spectrum of Mi field isolates by a variety of gene-based molecular techniques indicated a higher degree of genetic homogeneity than predicted on the basis of the phenotypic variability. All known strains of Mi were detected by the method developed. Together with an M. meleagridis-PCR test based on the same gene, the Multi-species PCR test is a highly valuable tool for diagnosis of pathogenic mycoplasmas in single or mixed infection. The further application of this rapid and specific test as a part of Mi and overall mycoplasma control programs will be dependent on developments in the turkey industry.

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