scholarly journals RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics.

2021 ◽  
Author(s):  
Michal Rozanski ◽  
Aurelia Walczak-Drzewiecka ◽  
Jolanta Witaszewska ◽  
Ewelina Wojcik ◽  
Arkadiusz Guzinski ◽  
...  
Keyword(s):  
Screening Test ◽  
False Negative ◽  
Clinical Samples ◽  
Highly Sensitive ◽  
In Vitro Diagnostic ◽  
Polymerase Chain ◽  
Asymptomatic Individuals ◽  
Pooled Samples ◽  
Rna Concentration

Swab, quantitative, reverse transcription polymerase chain reaction (RT-qPCR) tests remain the gold standard of diagnostics of SARS-CoV-2 infections. However, these tests are costly and time-consuming, and swabbing limits their throughput. We developed a 3-gene, seminested RT qPCR test with SYBR green-based detection, optimized for testing pooled saliva samples for high-throughput diagnostics of epidemic-affected populations. The proposed two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with the mentioned highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the in vitro diagnostic (IVD) test. The screening test was able to detect 5 copies of the viral genome in 10 μl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison (testing artificial pooled samples), the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of 4 revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with a fraction of the costs of individual RT-PCR testing.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Sample-Pooling Strategy for SARS-CoV-2 Detection among Students and Staff of the University of Sannio

Diagnostics ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1166
Author(s):  
Immacolata Polvere ◽  
Elena Silvestri ◽  
Lina Sabatino ◽  
Antonia Giacco ◽  
Stefania Iervolino ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Virus Detection ◽  
Testing Procedures ◽  
Stem Infection ◽  
In Vitro Diagnostic ◽  
Sample Pooling ◽  
Pooling Strategy ◽  
Asymptomatic Individuals ◽  
The University

Since the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, it has been clear that testing large groups of the population was the key to stem infection and prevent the effects of the coronavirus disease of 2019, mostly among sensitive patients. On the other hand, time and cost-sustainability of virus detection by molecular analysis such as reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) may be a major issue if testing is extended to large communities, mainly asymptomatic large communities. In this context, sample-pooling and test grouping could offer an effective solution. Here we report the screening on 1195 oral-nasopharyngeal swabs collected from students and staff of the Università degli Studi del Sannio (University of Sannio, Benevento, Campania, Italy) and analyzed by an in-house developed multiplex RT-qPCR for SARS-CoV-2 detection through a simple monodimensional sample pooling strategy. Overall, 400 distinct pools were generated and, within 24 h after swab collection, five positive samples were identified. Out of them, four were confirmed by using a commercially available kit suitable for in vitro diagnostic use (IVD). High accuracy, sensitivity and specificity were also determined by comparing our results with a reference IVD assay for all deconvoluted samples. Overall, we conducted 463 analyses instead of 1195, reducing testing resources by more than 60% without lengthening diagnosis time and without significant losses in sensitivity, suggesting that our strategy was successful in recognizing positive cases in a community of asymptomatic individuals with minor requirements of reagents and time when compared to normal testing procedures.

scholarly journals UTILITY OF ANTIGEN DETECTION TEST AND POLYMERASE CHAIN REACTION IN THE DIFFERENTIATION OF TUBERCULOUS AND NON-TUBERCULOUS MYCOBACTERIA

2017 ◽  
Vol 10 (6) ◽  
pp. 289
Author(s):  
Yogita Singh ◽  
Raji Vasanth ◽  
Shrikala Baliga ◽  
Dhanashree B
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Chain Reaction ◽  
College Hospital ◽  
Medical College ◽  
Negative Results ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Objectives: Cultivation and identification of mycobacteria to species level remains difficult and time-consuming. Hence, easy and rapid diagnostic methods are necessary for the differentiation of Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). The present study aims to detect and differentiate MTB from NTM isolated from clinical samples by immunochromatographic test (ICT) and polymerase chain reaction (PCR). Methods: Over a period of 1 year, clinical samples (n=496) received from suspected cases of TB, at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore were cultured to isolate Mycobacterium spp. Identification of all the isolates was done by conventional biochemical technique, ICT, and PCR. Results: Among the 496 samples processed, 49 (9.87%) were acid-fast bacilli smear positive and 59 (11.89%) samples showed the growth of Mycobacterium spp. Among these, 10 were rapid growers, 49 were slow-growing mycobacteria, out of which 30 were MTB as identified by conventional biochemical reaction. Out of 59 Mycobacterial isolates subjected to ICT for the detection of MPT 64 antigen, only 28 were identified as MTB. However, all the 30 isolates were correctly identified as MTB by PCR. Conclusion: Hence, PCR is essential for rapid differentiation of non-tuberculous Mycobacterium from MTB. False negative results seen with immunochromatographic MPT 64 antigen assay could be due to mutations within the mpt64 gene. Further studies are necessary to characterize these PCR-positive and immunochromatographic assay negative MTB isolates.

scholarly journals Multianalyte Tests for the Early Detection of Cancer: Speedbumps and Barriers

2007 ◽  
Vol 2 ◽  
pp. 117727190700200 ◽  
Author(s):  
Michael A. Tainsky ◽  
Madhumita Chatterjee ◽  
Nancy K. Levin ◽  
Sorin Draghici ◽  
Judith Abrams
Keyword(s):  
Early Detection ◽  
Tumor Antigens ◽  
Early Stage ◽  
False Negative ◽  
Clinical Intervention ◽  
Screening Tests ◽  
Molecular Event ◽  
Non Invasive ◽  
In Vitro Diagnostic

It has become very clear that a single molecular event is inadequate to accurately predict the biology (or pathophysiology) of cancer. Furthermore, using any single molecular event as a biomarker for the early detection of malignancy may not comprehensively identify the majority of individuals with that disease. Therefore, the fact that technologies have arisen that can simultaneously detect several, possibly hundreds, of biomarkers has propelled the field towards the development of multianalyte-based in vitro diagnostic early detection tests for cancer using body fluids such as serum, plasma, sputum, saliva, or urine. These multianalyte tests may be based on the detection of serum autoantibodies to tumor antigens, the presence of cancer-related proteins in serum, or the presence of tumor-specific genomic changes that appear in plasma as free DNA. The implementation of non-invasive diagnostic approaches to detect early stage cancer may provide the physician with evidence of cancer, but the question arises as to how the information will affect the pathway of clinical intervention. The confirmation of a positive result from an in vitro diagnostic cancer test may involve relatively invasive procedures to establish a true cancer diagnosis. If in vitro diagnostic tests are proven to be both specific, i.e. rarely produce false positive results due to unrelated conditions, and sufficiently sensitive, i.e. rarely produce false negative results, then such screening tests offer the potential for early detection and personalized therapeutics using multiple disease-related targets with convenient and non-invasive means. Here we discuss the technical and regulatory barriers inherent in development of clinical multianalyte biomarker assays.

scholarly journals Detection of LytA Genes in Streptococcus pneumoniae Isolated from sputum pneumonia patients

Biomedika ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 23-30
Author(s):  
Mustika Sari Hutabarat ◽  
Firdaus Hamid ◽  
Irawaty Djaharuddin ◽  
Alfian Zainuddin ◽  
Rossana Agus ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
False Negative ◽  
Pneumococcal Infection ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Streptococcus pneumoniae (pneumococcus) is a Gram-positive facultative anaerobic bacterium that is a major cause of morbidity and mortality worldwide. But the lack of reporting of disease by this bacterium in Indonesia, one of the causes is because the diagnosis of pneumococcal infection is often clinically not typical and conventional methods which are still the standard gold method often give false-negative results. So the purpose of this study was to evaluate the performance of culture and molecular diagnostic methods using the Polymerase Chain Reaction (PCR) technique in detecting Streptococcus pneumoniae in sputum clinical samples using the Autolysin (LytA) gene which is a virulence factor of this bacterium. 57 isolates from 60 samples were confirmed as Streptococcus sp through microscopic identification, culture, and biochemical tests. Then the sensitivity test with an optochin test of 9 (9%) compared the results descriptively with the PCR technique using the Autolysin A (LytA) gene which was obtained more sensitive by 15 (25%).

scholarly journals Påvisning av nye biomarkører i plasma-proteomet

2009 ◽  
Vol 16 (1) ◽  
Author(s):  
Jens P. Berg ◽  
John Henninge ◽  
Terje Lund
Keyword(s):  
Diagnostic Procedures ◽  
Prophylactic Surgery ◽  
Clinical Samples ◽  
Plasma Proteome ◽  
Post Translational Modifications ◽  
Novel Proteins ◽  
In Vitro Diagnostic ◽  
Close Cooperation ◽  
New Biomarkers

<p>Svar på laboratorieanalyser er av avgjørende betydning for ressursbruk i helsevesenet i forbindelse med utredning, behandling og oppfølgning av pasienter. Undersøkelser fra USA viser at ca. 70% av alle beslutninger som foretas i helsevesenet i stor grad er basert på resultater fra laboratoriene (1). Størst betydning for bruk av ressurser og folkehelse har laboratorieanalyser som kan brukes til å identifisere personer med økt risiko for sykdom som kan forebygges eller kureres. De mest dramatiske eksempler på dette finner man for noen dominant arvelige kreftformer hvor en enkelt genetisk undersøkelse kan påvise personer som trenger ekstra oppfølgning og i enkelte tilfeller profylaktisk kirurgisk behandling. Blod utgjør det viktigste materialet for laboratorieundersøkelser. I denne artikkelen vil vi beskrive noen av mulighetene som finnes i letingen etter nye biomarkører i form av proteiner og peptider i plasmaproteomet som betegner det totale uttrykket av proteiner i plasma. Utviklingen av immunometriske og massespektrometriske metoder har åpnet nye muligheter til å finne nye sykdomsmarkører både som ”nye” proteiner og i form av modifikasjoner som er dannet etter at proteinet er syntetisert (post-translasjonelle modifikasjoner). Effektiv testing av kombinasjoner av flere titalls markører gjør det mulig å lete etter sykdomsspesifikke ”fingeravtrykk” i proteomet. Plasma-proteomet karakteriseres imidlertid også av at bare noen få proteiner utgjør kvantitativt det meste av proteomet og av store konsentrasjonsforskjeller mellom de ulike proteinene. God tilgang på godt karakteriserte kliniske materialer er helt nødvendig for å kunne validere nytten av en ny biomarkør. Et effektivt samarbeid mellom metode-utviklere og biobanker er derfor av avgjørende betydning for hvor raskt en ny biomarkør kan bli en diagnostisk eller prognostisk faktor.</p><p><em>Determination of new biomarkers in the plasma proteome.</em> Decision making and use of health care resources depend to a large extent on data from in vitro diagnostic procedures. Laboratory results may have its largest impact on diseases that can be prevented or cured. Most dramatically a single genetic analysis can in certain dominantly inherited diseases differentiate between subjects who are at high risk of developing cancer and subjects who have a risk comparable to the general population. Ultimately, the test may identify individuals eligible for curative prophylactic surgery. Blood is the most important specimen for laboratory investigations. This paper describes some of the possibilities that are emerging in the search for new protein and peptide biomarkers in the plasma proteome, which is the total expression of proteins in plasma. The development of new immunometric and mass spectrometric methods has opened new strategies to identify disease markers both as novel proteins and differences in their post-translational modifications. Efficient testing of combinations of tens of markers simultaneously makes it technically possible to search for fingerprints of specific diseases in the proteome. However, a few plasma proteins dominate quantitatively and there are huge concentration differences among the proteins. Access to well characterized clinical samples is of great importance for the validation of new candidate markers. Close cooperation between biomarker hunters and serum or plasma biobanks is a crucial determinant for the transition of a biomarker candidate to a potential diagnostic of prognostic factor</p>

Sensitivity evaluation of the Gen-Probe AMP-CT assay by pooling urine samples for the screening of Chlamydia trachomatis urogenital infection

2002 ◽  
Vol 13 (8) ◽  
pp. 540-542 ◽  
Author(s):  
J P Gomes ◽  
S Viegas ◽  
A Paulino ◽  
M A Catry
Keyword(s):  
Chlamydia Trachomatis ◽  
Population Based ◽  
Pcr Assay ◽  
Urogenital Infection ◽  
Sensitivity Evaluation ◽  
Highly Sensitive ◽  
Polymerase Chain ◽  
Pooled Samples ◽  
Urine Pool ◽  
Urine Specimens

The sensitivity of two urine pool sizes versus individual testing, to detect Chlamydia trachomatis urogenital infection, was evaluated using the Gen-Probe AMP-CT assay. Thirty-three (33) known polymerase chain reaction (PCR) positive urine specimens were combined with 231 fresh first-catch urine (FCU) samples in 33 groups of four and 33 groups of eight, to make up 4X and 8X pooled samples, respectively. Gen-Probe AMP-CT assay was performed on pools as well as on individual samples at the same time. For the discrepant cases, the known positive samples were diluted 1:4 and 1:8 using the manufacturer's dilution buffer and were retested. Additional positive specimens found among fresh FCU samples were also tested by the Amplicor-PCR assay to confirm their positivity. The sensitivities of 8X pooling, 4X pooling and individual testing were 86.5%, 94.3% and 91.9%, respectively. The Gen-Probe AMP-CT assay applied to a 4X urine pooling model was highly sensitive and may be useful for a population based screening programme.

scholarly journals The Use of a Mimic to Detect Polymerase Chain Reaction– Inhibitory Factors in Feces Examined for the Presence of Lawsonia Intracellularis

2003 ◽  
Vol 15 (3) ◽  
pp. 268-273 ◽  
Author(s):  
Magdalena Jacobson ◽  
Stina Englund ◽  
András Ballagi-Pordány
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Clinical Samples ◽  
Lawsonia Intracellularis ◽  
Chain Reaction ◽  
Fecal Samples ◽  
Wild Boars ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors were present. When fecal samples were spiked with the mimic, the detection limit was 102 molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 103 mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR.

Verification of clinical samples, positive in AMPLICOR Neisseria gonorrhoeae polymerase chain reaction, by 16S rRNA and gyrA compared with culture

2005 ◽  
Vol 16 (6) ◽  
pp. 415-419 ◽  
Author(s):  
Åsa Airell ◽  
Emma Lindbäck ◽  
Ferda Ataker ◽  
Kirsti Jalakas Pörnull ◽  
Bengt Wretlind
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Neisseria Gonorrhoeae ◽  
False Negative ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Gyra Gene ◽  
Two Samples ◽  
Polymerase Chain

We compared 956 samples for AMPLICOR Neisseria gonorrhoeae polymerase chain reaction (PCR) (Roche) with species verification using the 16S rRNA gene to verification using gyrA gene. Control was the culture method. The gyrA verification uses pyrosequencing of the quinolone resistance-determining region of gyrA. Of 52 samples with optical density ≥0.2 in PCR, 27 were negative in culture, two samples from pharynx were false negative in culture and four samples from pharynx were false positives in verification with 16S rRNA. Twenty-five samples showed growth of gonococci, 18 of the corresponding PCR samples were verified by both methods; three urine samples were positive only in gyrA ; and one pharynx specimen was positive only in 16S rRNA. Three samples were lost. We conclude that AMPLICOR N. gonorrhoeae PCR with verification in gyrA gene can be considered as a diagnostic tool in populations with low prevalence of gonorrhoea and that pharynx specimens should not be analysed by PCR.

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