scholarly journals OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1314
Author(s):  
Eduarda Carvalho-Correia ◽  
Carla Calçada ◽  
Fernando Branca ◽  
Nuria Estévez-Gómez ◽  
Loretta De Chiara ◽  
...  
Keyword(s):  
Cross Reactivity ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
S Gene ◽  
Genomic Knowledge ◽  
Gene Assay ◽  
One Step

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide–sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

scholarly journals Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay

2021 ◽  
Vol 11 ◽  
Author(s):  
Suhua Xin ◽  
Hong Zhu ◽  
Chenglin Tao ◽  
Beibei Zhang ◽  
Lan Yao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epidemiologic Study ◽  
Clinical Diagnostics ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Chicken Farm

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

scholarly journals Reverse transcription loop-mediated isothermal amplification combined with nanoparticles-based biosensor for diagnosis of COVID-19

2020 ◽  
Author(s):  
Xiong Zhu ◽  
Xiaoxia Wang ◽  
Limei Han ◽  
Ting Chen ◽  
Licheng Wang ◽  
...  
Keyword(s):  
Public Health ◽  
Reverse Transcription ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Public Health Issue ◽  
Analytical Sensitivity ◽  
Sample Collection ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

ABSTRACTGiven the scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, known as 2019-nCov) infection (COVID-19), the ongoing global SARS-CoV-2 outbreak has become a huge public health issue. Rapid and precise diagnostic methods are thus immediately needed for diagnosing COVID-19, providing timely treatment and facilitating infection control. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with nanoparticles-based biosensor (NBS) assay (RT-LAMP-NBS) was successfully established for rapidly and accurately diagnosing COVID-19. A simple equipment (such as heating block) was required for maintaining a constant temperature (63°C) for only 40 min. Using two designed LAMP primer sets, F1ab (opening reading frame 1a/b) and np (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified and detected in a ‘one-step’ and ‘single-tube’ reaction, and the detection results were easily interpreted by NBS. The sensitivity of SARS-CoV-2 RT-LAMP-NBS was 12 copies (each of detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. Among clinically diagnosed COVID-19 patients, the analytical sensitivity of SARS-CoV-2 was 100% (33/33) in the oropharynx swab samples, and the assay’s specificity was also 100% (96/96) when analyzed the clinical samples collected from non-COVID-19 patients. The total diagnosis test from sample collection to result interpretation only takes approximately 1 h. In sum, the RT-LAMP-NBS is a promising tool for diagnosing the current SARS-CoV-2 infection in first line field, public health and clinical laboratories, especially for resource-challenged regions.

A point-of-care diagnostic for differentiating Ebola from endemic febrile diseases

2018 ◽  
Vol 10 (471) ◽  
pp. eaat0944 ◽  
Author(s):  
David Sebba ◽  
Alexander G. Lastovich ◽  
Melody Kuroda ◽  
Eric Fallows ◽  
Joshua Johnson ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Clinical Symptoms ◽  
Point Of Care ◽  
Hemorrhagic Fever ◽  
Diagnostic Methods ◽  
Outbreak Detection ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Live Virus ◽  
And Control

Hemorrhagic fever outbreaks such as Ebola are difficult to detect and control because of the lack of low-cost, easily deployable diagnostics and because initial clinical symptoms mimic other endemic diseases such as malaria. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need. Although rapid tests such as lateral flow can be broadly deployed, they are typically not well-suited for differentiating among multiple diseases presenting with similar symptoms. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect and differentiate infection with Ebola virus from other more common febrile diseases. Here, we developed and tested an immunoassay technology that uses surface-enhanced Raman scattering (SERS) tags to simultaneously detect antigens from Ebola, Lassa, and malaria within a single blood sample. Results are provided in <30 min for individual or batched samples. Using 190 clinical samples collected from the 2014 West African Ebola outbreak, along with 163 malaria positives and 233 negative controls, we demonstrated Ebola detection with 90.0% sensitivity and 97.9% specificity and malaria detection with 100.0% sensitivity and 99.6% specificity. These results, along with corresponding live virus and nonhuman primate testing of an Ebola, Lassa, and malaria 3-plex assay, indicate the potential of the SERS technology as an important tool for outbreak detection and clinical triage in low-resource settings.

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 A New Molecular Detection System for Canine Distemper Virus Based on a Double-Check Strategy

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1632
Author(s):  
Sabrina Halecker ◽  
Sabine Bock ◽  
Martin Beer ◽  
Bernd Hoffmann
Keyword(s):  
Canine Distemper Virus ◽  
Parallel Implementation ◽  
Detection System ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Wild Animals ◽  
Canine Distemper ◽  
One Step ◽  
Diagnostic Sensitivity And Specificity

Due to changing distemper issues worldwide and to inadequate results of an inter-laboratory study in Germany, it seems sensible to adapt and optimize the diagnostic methods for the detection of the canine distemper virus (CDV) to the new genetic diversity of virus strains. The goal of the project was the development, establishment and validation of two independent one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) methods for the safe detection of CDV in domestic and wild animals. For this purpose, an existing CDV-RT-qPCR was decisively adapted and, in addition, a completely new system was developed. Both CDV-RT-qPCR systems are characterized by a very high, comparable analytical and diagnostic sensitivity and specificity and can be mutually combined with inhibition or extraction controls. The reduction in the master mix used allows for the parallel implementation of both CDV-RT-qPCR systems without significant cost increases. For validation of the new CDV-RT-qPCR duplex assays, a panel comprising 378 samples derived from Germany, several European countries and one African country were tested. A sensitivity of 98.9% and a specificity of 100% were computed for the new assays, thus being a reliable molecular diagnostic tool for the detection of CDV in domestic and wild animals.

scholarly journals Development of an Improved Whole Blood Assay for Diagnosis of Latent and Active Tuberculosis Cases

2021 ◽  
pp. 197-209
Author(s):  
Rupam R. Nashine ◽  
Amit R. Nayak ◽  
Aliabbas Husain ◽  
Gargi D. Mudey ◽  
Hatim F. Daginawala ◽  
...  
Keyword(s):  
Whole Blood ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Interferon Gamma Release Assay ◽  
Antigenic Peptides ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Variable Sensitivity ◽  
Latent Tb Infection

Background: Latent TB infection (LTBI) is an infection where the presence of disease causing organism M. tuberculosis is there without any sign and symptoms of the disease hence mostly remains undiagnosed, though Tuberculin skin test (TST) and Interferon Gamma Release Assay (IGRA) were used to diagnose the LTBI. They have their limitations, TST gives major cross-reactivity with BCG vaccine and gives inaccurate results in individuals who have taken BCG and IGRA are very costly and variable sensitivity is repeated in various populations hence the modifications are needed in the IGRA for proper diagnosis of LTBI. Objectives: In the proposed study we aimed to develop an improved whole blood assay                    towards a diagnosis of latent and active TB infection as an alternative to the Quantiferon QFT assay Methodology: Synthetic antigenic peptides against latency specific antigens will be designed and synthesized. Designed peptides will be screened for LTBI specific cytokine by in-vitro experiments. Development & production of Whole assay using selected peptides evaluation of developed assay through ELISA in clinical samples. Expected Results: Latent specific peptides will be identified and peptide-based whole blood assay for detection and diagnosis of tuberculosis will be developed as an indigenous alternative for the existing QFT assay. Conclusion: An improved whole blood assay will be developed for screening of LTBI in the Indian population.

scholarly journals Comparison of commercial RT-PCR diagnostic kits for COVID-19

2020 ◽  
Author(s):  
Puck B. van Kasteren ◽  
Bas van der Veer ◽  
Sharon van den Brink ◽  
Lisa Wijsman ◽  
Jørgen de Jonge ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Cross Reactivity ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Specific Method ◽  
Preliminary Evaluation ◽  
Rt Pcr ◽  
Accurate Identification ◽  
Clinical Sensitivity ◽  
Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

scholarly journals Development of a multiplexed qPCRs-based approach for the diagnosis of Dirofilaria immitis, D. repens, Acanthocheilonema reconditum and the others filariosis

2019 ◽  
Author(s):  
Younes Laidoudi ◽  
Bernard Davoust ◽  
Marie Varloud ◽  
El Hadji Amadou Niang ◽  
Florence Fenollar ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Dirofilaria Immitis ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Typing Method ◽  
Filarial Nematodes ◽  
Acanthocheilonema Reconditum ◽  
Kappa Agreement

AbstractBackgroundThe frequent canine filariosis are caused by zoonotic filarial nematodes called Dirofilaria immitis, D. repens and Acanthocheilonema reconditum (Spirurida: Onchocercidae). The absence of reliable diagnostic tools to diagnose and discriminate between these infections as well as their different forms constitutes a major obstacle to their control. The serological diagnosis of heartworm disease has recently shown both sensitivity and specificity problems.FindingsHerein, we developed and set up a novel molecular approach for an improved detection of the occult and non-occult filarioses, especially those caused by A. reconditum, D. immitis and D. repens as well as their differential diagnosis based on qPCRs assays. This approach designated the “Combined multiplex approach”, proceeds as follows: Filaria and wolbachia identification using the newly customized 28S-based pan-filarial and 16S-based pan-wolbachia qPCRs, respectively, followed by the fast typing method of positive samples using the triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and that of D. repens. The analytical sensitivity of the newly qPCR systems was confirmed by the detection limit of wolbachia and filaria DNA ranged from 5E-1 to 1.5E-4 mf/ml of blood with an R² higher than 0.99, Cohen’s Kappa agreement ranged from 0.98 to 1. The approach was complemented by a pan-filarial COI and pan-Wolbachia ftsZ PCR for the identification of other filarial parasites and their Wolbachia, respectively.When tested on clinical samples, the results are as follows: 29.2 % (49/168) tested positive to at least filariae or wolbachiae DNA. 19 samples of them tested positive for filarial DNA, 9 for wolbachia DNA and 21 for both. Filarial species and Wolbachia genotype were also identified by the combined multiplex approach from all the positive samples. The single DNA of D. immitis was identified in 12 samples, D. repens in 7, and A. reconditum in 15 samples, the co-infection was observed in 5 samples, 4 for both Dirofilaria and one harbored the three species. Therefore, 22 samples were positive for Wolbachia endosymbiont of D. immitis, 3 for that of D. repens and 5 for both genotypes. A newly duplex qPCR developed for the differential diagnosis of heartworm and French heartworm (Angiostrongylus vasorum) was successfully validated in vitro. However, no DNA of this latter was detected in canine blood samples used in this study. The immunochromatographic test for dirofilariasis antigen during evaluation before and after thermal pretreatment of sera showed substantial agreement (K=0.6) and weak agreement (K=0.15), respectively.ConclusionThe proposed molecular tool targeting filarial genes and associated Wolbachia genes is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariasis. We believe that the current diagnosis of heartworm based on antigen detection should be always confirmed by qPCR-based essays; the heat-pretreatment of sera is useless and strongly discouraged.

scholarly journals Evaluation of an Antigen Detection Rapid Diagnostic Test for Detection of SARS-CoV-2 in Clinical Samples

COVID ◽  
2021 ◽  
Vol 1 (4) ◽  
pp. 775-783
Author(s):  
Hoi-Ying Lam ◽  
Ka-Yi Leung ◽  
Ruiqi Zhang ◽  
Danlei Liu ◽  
Yujing Fan ◽  
...  
Keyword(s):  
Viral Load ◽  
Diagnostic Test ◽  
Rapid Diagnostic Test ◽  
Large Scale ◽  
Cross Reactivity ◽  
Antigen Detection ◽  
Youden Index ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Large Scale Screening

Antigen detection rapid diagnostic tests have been developed for first-line large-scale screening given their rapidity, simplicity, and accuracy. This study evaluates the diagnostic performance of an antigen detection rapid diagnostic test (BLOK BioScience, London, UK) detecting SARS-CoV-2 nucleocapsid protein. Serially diluted SARS-CoV-2 isolate and 110 NPS from COVID-19 patients were tested to determine the test’s sensitivity, and other viral isolates and 20 NPS from non-infected individuals were, for specificity, also tested. Ten clinical samples from COVID-19 patients with SARS-CoV-2 variants, including alpha, beta, gamma, delta, and eta variants, were collected to evaluate the test’s potential application in detecting emerging variants. Overall sensitivity was 92%, and stratifying into viral loads yielded 100% for Ct < 25 samples including SARS-CoV-2 variants, but 11.11% for Ct ≥ 30 samples. The analytical sensitivity of log10 TCID50/mL 2.0 was identified for SARS-CoV-2. Ninety-seven percent specificity with only SARS-CoV cross-reactivity lead to the Youden index of 0.89. The rapid diagnostic test has a high sensitivity for detecting SARS-CoV-2 in high viral load samples, possibly including emerging SARS-CoV-2 variants, but reduced sensitivity in low viral load samples suggests its optimized usage as a complementary testing method to other tests, including RT-PCR or a point-of-care test for large-scale screening, particularly for pandemic areas or airport border infection control.

Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction

2019 ◽  
Vol 15 (7) ◽  
pp. 1598-1608
Author(s):  
Hongna Liu ◽  
Kathryn Heflin ◽  
Jian Han ◽  
Matt Conover ◽  
Leslie Wagner ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Bacterial Species ◽  
Bloodstream Infections ◽  
Cross Reactivity ◽  
Blood Cultures ◽  
Molecular Diagnostic ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
High Level

We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, in vitro diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 105–1.7 × 107 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.

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