scholarly journals ASSESSMENT OF POTENTIAL SARS-CoV-2 VIRUS N GENE INTEGRATION INTO HUMAN GENOME REVEALS NO SIGNIFICANT IMPACT ON RT-qPCR COVID-19 DIAGNOSTIC TESTING

2021 ◽  
Author(s):  
Erica Briggs ◽  
William Ward ◽  
Sol Rey ◽  
Dylan Law ◽  
Katherine Nelson ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Clinical Laboratory ◽  
Diagnostic Testing ◽  
Scale Up ◽  
Mobile Element ◽  
N Gene ◽  
Small Subset ◽  
Gene Integration ◽  
Diagnostic Capabilities ◽  
Prime End

The SARS Coronavirus 2 (SARS-CoV-2) pandemic presents new scientific and scale-up challenges for diagnostic capabilities worldwide. The gold standard diagnostic for SARS-CoV-2 infection is a reverse transcription/quantitative PCR (RT-qPCR) which targets the viral genome, an assay that has now been performed on millions of patient specimens worldwide regardless of symptomatic status. Recently Zhang et al. suggested the possibility that the SARS-CoV-2 N gene could integrate into host cell DNA through the action of the LINE-1 retrotransposon, a mobile element that is potentially active in human somatic cells, thereby calling into question the veracity of N-gene based RT-qPCR for detection of SARS-CoV-2 infection. Accordingly, we assessed the potential impact of these purported integration events on nasal swab specimens tested at our clinical laboratory. Using an N-gene based RT-qPCR assay, we tested 768 arbitrarily selected specimens and identified 2 samples which resulted in a positive detection of viral sequence in the absence of reverse transcriptase, a necessary but not sufficient signal consistent with possible integration of the SARS-CoV-2 N gene into the host genome. Regardless of possible viral N gene integration into the genome, in this small subset of samples, all patients were still positive for SARS-CoV-2 infection, as indicated by a much lower Ct value for reactions performed in the presence of reverse transcriptase (RT) versus reactions performed without RT. Moreover, one of the two positives observed in the absence of RT also tested positive when using primers targeting ORF1ab, a gene closer to the 5 prime end of the genome. These data are inconsistent with the N gene integration hypothesis suggested by the studies by Zhang et al., and importantly, our results suggest little to no practical impact of possible SARS-CoV-2 genome integration events on RT-qPCR testing.

Clinical Laboratory and Diagnostic Testing

1991 ◽  
Vol 3 (3) ◽  
pp. 343-345
Author(s):  
Richard B. Rosse
Keyword(s):  
Clinical Laboratory ◽  
Diagnostic Testing

scholarly journals Development of dried tube specimens for Xpert MTB/RIF proficiency testing

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Kyle DeGruy ◽  
Katherine Klein ◽  
Zilma Rey ◽  
Patricia Hall ◽  
Andrea Kim ◽  
...  
Keyword(s):  
Proficiency Testing ◽  
Diagnostic Testing ◽  
Scale Up ◽  
The United States ◽  
Quality Assurance Programme ◽  
Continuous Quality ◽  
Resource Limited ◽  
Control And Prevention ◽  
Improvement Programme ◽  
Validation Testing

Background: Proficiency testing (PT) is part of a comprehensive quality assurance programme, which is critical to ensuring patients receive accurate and reliable diagnostic testing. Implementation of the Cepheid Xpert® MTB/RIF assay to aid in the diagnosis of tuberculosis has expanded rapidly in recent years; however, PT material for Xpert MTB/RIF is not readily available in many resource-limited settings.Objective: To develop an accurate and precise PT material based on the dried tube specimen (DTS) method, using supplies and reagents available in most tuberculosis culture laboratories.Methods: Dried tube specimens were produced at the United States Centers for Disease Control and Prevention from 2013 to 2015 by inactivating liquid cultures of well-characterised mycobacterial strains. Ten percent of DTS produced were tested with Xpert MTB/RIF and evaluated for accuracy and precision.Results: Validation testing across eight rounds of PT demonstrated that DTS are highly accurate, achieving an average of 96.8% concordance with the Xpert MTB/RIF results from the original mycobacterial strains. Dried tube specimen testing was also precise, with cycle threshold standard deviations below two cycles when inherent test cartridge variability was low.Conclusion: Dried tube specimens can be produced using equipment already present in tuberculosis culture laboratories, making Xpert MTB/RIF PT scale-up more feasible in resource-limited settings. Use of DTS may fill the gap in tuberculosis laboratory access to external quality assessment, which is an essential component of a comprehensive continuous quality improvement programme.

Nonparametric Decision Support Systems in Medical Diagnosis

2011 ◽  
pp. 1483-1500
Author(s):  
Steven Walczak ◽  
Bradley B. Brimhall ◽  
Jerry B. Lefkowitz
Keyword(s):  
Medical Diagnosis ◽  
Laboratory Tests ◽  
Clinical Laboratory ◽  
Predictive Performance ◽  
Diagnostic Model ◽  
Medical Problems ◽  
Diagnostic Models ◽  
Trained Neural Network ◽  
Diagnostic Capabilities ◽  
D Dimer

Patients face a multitude of diseases, trauma, and related medical problems that are difficult to diagnose and have large treatment and diagnostic direct costs, including pulmonary embolism (PE), which has mortality rates as high as 10%. Advanced decision-making tools, such as nonparametric neural networks (NN), may improve diagnostic capabilities for these problematic medical conditions. The research develops a backpropagation trained neural network diagnostic model to predict the occurrence of PE. Laboratory database values for 292 patients who were determined to be at risk for PE, with almost 15% suffering a confirmed PE, were collected and used to evaluate various NN models’ performances. Results indicate that using NN diagnostic models enables the leveraging of knowledge gained from standard clinical laboratory tests, specifically the d-dimer assay and reactive glucose, significantly improving overall positive predictive value, compared to using either test in isolation, and also increasing negative predictive performance.

Nonparametric Decision Support Systems in Medical Diagnosis

2011 ◽  
pp. 562-579
Author(s):  
Steven Walczak ◽  
Bradley B. Brimhall ◽  
Jerry B. Lefkowitz
Keyword(s):  
Medical Diagnosis ◽  
Laboratory Tests ◽  
Clinical Laboratory ◽  
Predictive Performance ◽  
Diagnostic Model ◽  
Medical Problems ◽  
Diagnostic Models ◽  
Trained Neural Network ◽  
Diagnostic Capabilities ◽  
D Dimer

Patients face a multitude of diseases, trauma, and related medical problems that are difficult to diagnose and have large treatment and diagnostic direct costs, including pulmonary embolism (PE), which has mortality rates as high as 10%. Advanced decision-making tools, such as nonparametric neural networks (NN), may improve diagnostic capabilities for these problematic medical conditions. The research develops a backpropagation trained neural network diagnostic model to predict the occurrence of PE. Laboratory database values for 292 patients who were determined to be at risk for PE, with almost 15% suffering a confirmed PE, were collected and used to evaluate various NN models’ performances. Results indicate that using NN diagnostic models enables the leveraging of knowledge gained from standard clinical laboratory tests, specifically the d-dimer assay and reactive glucose, significantly improving overall positive predictive value, compared to using either test in isolation, and also increasing negative predictive performance.

Laboratory Medicine and Diagnostic Testing

2004 ◽  
Vol 94 (2) ◽  
pp. 194-197
Author(s):  
Noubar Kessimian
Keyword(s):  
Medical Practice ◽  
Clinical Laboratory ◽  
Diagnostic Testing ◽  
Laboratory Data ◽  
Laboratory Medicine ◽  
Test Results ◽  
Vital Component ◽  
Sensitivity Specificity

The clinical laboratory is a vital component of modern podiatric medical practice. In order to interpret laboratory data correctly, the practitioner must understand the essentials of diagnostic testing. These essentials include precision, accuracy, sensitivity, specificity, and prevalence-based values of a given test. In addition, the podiatric physician should be aware of the limitations, variations, and interferences that can spuriously alter test results. (J Am Podiatr Med Assoc 94(2): 194-197, 2004)

Clinical Laboratory and Diagnostic Testing

1990 ◽  
Vol 2 (2) ◽  
pp. 223-224
Author(s):  
Richard B. Rosse
Keyword(s):  
Clinical Laboratory ◽  
Diagnostic Testing

scholarly journals Development of an assay for the detection and quantification of the measles virus nucleoprotein (N) gene using real-time reverse transcriptase PCR

2009 ◽  
Vol 58 (5) ◽  
pp. 638-643 ◽  
Author(s):  
Miho Akiyama ◽  
Hirokazu Kimura ◽  
Hiroyuki Tsukagoshi ◽  
Katsuya Taira ◽  
Katsumi Mizuta ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Measles Virus ◽  
N Gene ◽  
Reverse Transcriptase Pcr ◽  
Quantification Method ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Nucleoprotein N ◽  
Detection And Quantification

We developed a new quantification method for the measles virus (MeV) nucleoprotein (N) gene using real-time reverse transcriptase PCR. This method allowed us to quantify 101–107 copies per reaction (corresponding to 5×10−1–5×105 copies μl−1) of the MeV N gene. We also quantified the MeV N gene from the throat swabs of 22 patients with measles as well as the MeV genotypes A, D3, D5, D9 and H1 in viral suspensions derived from MeV-infected cells. As a result, 3.9×103–5.2×106 copies ml−1 and 7.4×107–2.0×108 copies ml−1 of the MeV genomes (N gene) were detected in the throat swabs and viral suspensions, respectively. No other viruses (enteroviruses, respiratory syncytial virus, human metapneumovirus or mumps virus) were detected in the assay. The results suggest that this method is applicable to the detection and quantification of some genotypes of MeV.

scholarly journals Rapid, sensitive and specific SARS coronavirus-2 detection: a multi-center comparison between standard qRT-PCR and CRISPR based DETECTR.

2020 ◽  
Author(s):  
Eelke Brandsma ◽  
Han JMP Verhagen ◽  
Thijs J.W. van de Laar ◽  
Eric C.J. Claas ◽  
Marion Cornelissen ◽  
...  
Keyword(s):  
Point Of Care ◽  
Diagnostic Testing ◽  
Added Value ◽  
N Gene ◽  
Analytical Sensitivity ◽  
Qrt Pcr ◽  
Negative Results ◽  
Point Of Care Test ◽  
Ribonucleoprotein Complexes ◽  
Large Patient

Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of isothermal reverse transcriptase loop mediated amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to qRT-PCR without sacrificing sensitivity/specificity. Here we compare qRT-PCR with DETECTR to diagnose COVID-19 on 378 patient samples and report a 95% reproducibility. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared to qRT-PCR, however, this was not confirmed in a large patient cohort. The data showed that both techniques are equally sensitive in detecting SARS-CoV-2 providing an added value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different gRNAs can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point of care test (POCT), showed a 100% correlation to the high-throughput DETECTR assay. Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other human coronaviruses. As there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR-platforms for routine non- SARS-CoV-2 diagnostic testing.

scholarly journals Practical diagnostic testing for human immunodeficiency virus.

1988 ◽  
Vol 1 (1) ◽  
pp. 124-138 ◽  
Author(s):  
J B Jackson ◽  
H H Balfour
Keyword(s):  
Human Immunodeficiency Virus ◽  
Western Blot ◽  
Reverse Transcriptase ◽  
Indirect Immunofluorescence ◽  
Diagnostic Testing ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hiv Reverse Transcriptase ◽  
Reverse Transcriptase Enzyme ◽  
Immunodeficiency Virus ◽  
Hiv Antigen

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures.

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