scholarly journals Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 RNA Detection

2021 ◽  
Author(s):  
Shuang Wu ◽  
Xiaolu Shi ◽  
Qiongcheng Chen ◽  
Yixiang Jiang ◽  
Le Zuo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Comparative Study ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Predictive Values ◽  
Health Crisis ◽  
Sensitivity Specificity

Abstract Background: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy. Methods: Fifty-two clinical samples were used including throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising confirmed (n=26) and negative cases (n=26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. Results: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa≥0.75); Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all kits were statistically significant (P<0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. Conclusions: This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic. Keywords: SARS-CoV-2; COVID-19; nucleic acid detection; real-time reverse transcriptase PCR (RT-qPCR); cross-priming isothermal amplification (CPA)

scholarly journals Comparative evaluation of six nucleic acid amplification kits for SARS-CoV-2 RNA detection

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Shuang Wu ◽  
Xiaolu Shi ◽  
Qiongcheng Chen ◽  
Yixiang Jiang ◽  
Le Zuo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Comparative Study ◽  
Clinical Performance ◽  
Positive Sample ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Predictive Values ◽  
Health Crisis ◽  
Specificity And Sensitivity ◽  
Sensitivity Specificity

Abstract Background SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy. Methods Fifty-two clinical samples were used including throat swabs (n = 30), nasal swabs (n = 7), nasopharyngeal swabs (n = 7) and sputum specimens (n = 8), comprising confirmed (n = 26) and negative cases (n = 26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. Results For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa ≥ 0.75); Kits D and E were less congruent (0.4 ≤ Kappa < 0.75). Differences between all kits were statistically significant (P < 0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. Conclusions This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.

scholarly journals Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 Detection

2020 ◽  
Author(s):  
Shuang Wu ◽  
Xiaolu Shi ◽  
Qiongcheng Chen ◽  
Yixiang Jiang ◽  
Le Zuo ◽  
...  
Keyword(s):  
Comparative Study ◽  
Clinical Diagnosis ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Predictive Values ◽  
Health Crisis ◽  
Pcr Assays ◽  
Sensitivity Specificity

Abstract SARS-CoV-2 is a newly emerged coronavirus that was isolated from human infections in recent months. Since drugs and vaccines of Covid-19 are still being developed, accurate pathogen detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been reliably used for the detection and confirmation of SARS-CoV-2 since the beginning of outbreak, whereas isothermal nucleic acid amplification based point of care automated assays has also been considered as a simpler and rapid alternative. However, since these assays have only been developed and applied for clinical use within a short timeframe, their analytical performance has not been adequately compared to-date. We describe a comparative study between a newly developed cross primer isothermal amplification (CPA) assay (Kit A) and five RT-PCR assays (Kits B to F), using clinical diagnosis as the reference standard to evaluate their sensitivity, specificity, predictive values and accuracy analysis. Clinical samples used (n=52) included throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprised of positive (n=26) and cleared cases (n=26) by clinical diagnosis. For the CPA assay (Kit A), the sensitivity, specificity, positive and negative predictive values and accuracy were 100%. Among the five RT-PCR kits, Kits B, C and F had good agreement with clinical diagnosis (Kappa≥0.75), Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all assays were statistically significant (P<0.001). The reproducibility of RT-PCR assays was determined using a positive sample that was verified by all assays, with standard deviations (SD) between 0.35 and 0.87, and coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. To further evaluate the CPA assay (Kit A) compared to Kits B and F, throat swabs from close contacts of cases (n=200) were analyzed. All three kits identified the same positive samples and showed total agreement. This is the first comparative study to evaluate a CPA assay and RT-PCR assays for SARS-CoV-2 detection, which could serve as a reference for clinical laboratories and inform testing protocols amid the rapidly evolving COVID-19 pandemic.

scholarly journals Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples

2006 ◽  
Vol 52 (4) ◽  
pp. 624-633 ◽  
Author(s):  
Jin Li ◽  
Fengfei Wang ◽  
Harvey Mamon ◽  
Matthew H Kulke ◽  
Lyndsay Harris ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genetic Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Pcr Primers ◽  
Medical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Correct Identification

Abstract Background: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. Methods: We modified one of the 2 PCR primers by use of an oligonucleotide “tail” fluorescently labeled at the 5′ end. An oligonucleotide complementary to this tail, carrying a 3′ quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. Results: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r2 &gt;0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. Conclusion: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.

scholarly journals Strategies for Isothermal Amplification of Nucleic Acids: are they Ready to be Applied in Point of Care Diagnosis of Mycosis?

2020 ◽  
Vol 11 (3) ◽  
pp. 10559-10571
Keyword(s):  
Nucleic Acid ◽  
Clinical Laboratory ◽  
Multiple Displacement Amplification ◽  
Rolling Circle Amplification ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Rolling Circle ◽  
Isothermal Nucleic Acid Amplification

The early detection of invasive fungal infection (IFD) is significant in order to decrease mortality in susceptible patients. There is, therefore, a need for sensitive and specific fungal species detection assays in a clinical laboratory for early targeted therapy. The isothermal amplification method may be useful for the screening of fungal isolates, especially in resource-poor settings. Therefore, our aim was to review the isothermal nucleic acid amplification methods and their applications in fungal pathogen detection. Out of 50 reported studies, 28, 12, 6, 2, and 2 studies used the isothermal-based assays of a loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and polymerase Spiral Reaction (PSR), respectively. Thirty-two studies used clinical samples, 18 pure culture, and four environmental samples. The diagnostic accuracy of isothermal nucleic acid amplification testing for pathogenic fungal was reported as high (sensitivity 0.89–1.0 and specificity 0.63–1.0) in all studies irrespective of the sample tested. Although the isothermal-based assays showed high sensitivity and specificity in reported studies, it is still poorer than that of PCR assays. However, improving the assay to make it simpler, more effective, and inexpensive compared with newer PCR methods are still needed.

scholarly journals A 30-minute nucleic acid amplification point-of-care test for genitalChlamydia trachomatisinfection in women: a prospective, multi-centre study of diagnostic accuracy

2017 ◽  
Author(s):  
EM Harding-Esch ◽  
EC Cousins ◽  
S-LC Chow ◽  
LT Phillips ◽  
CL Hall ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Accuracy ◽  
Clinical Pathways ◽  
Point Of Care ◽  
Specific Treatment ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Multi Centre Study ◽  
Predictive Values ◽  
Significant Difference

ABSTRACTBackgroundRapid Point-Of-Care Tests (POCTs) forChlamydia trachomatis(CT) may reduce onward transmission and reproductive sexual health (RSH) sequelae by reducing turnaround times between diagnosis and treatment. The io®single module system (Atlas Genetics Ltd) runs clinical samples through a microfluidic CT cartridge, delivering results in 30 minutes. We evaluated its performance on female genital samples in four UK Genito-Urinary Medicine (GUM)/RSH clinics.MethodsProspective diagnostic accuracy study, using BD ProbeTec CT/GC assay as the routine clinic nucleic acid amplification test (NAAT) as the initial comparator test, and the QIAgen Artus CT assay to resolve discrepancies. In these instances, the reference standard was defined as the resolved result when two out of three assay results concurred. Female participants aged ≥16 provided additional-to-routine self-collected vulvovaginal swabs. Samples were tested fresh with the io®CT assay within 7 days of collection, or were frozen at −80°C for later testing. Participant clinical, demographic and behavioural characteristics were collected to assess risk factors associated with CT infection.ResultsOf 785 participants recruited, final analyses were conducted on 709 (90.3%). CT prevalence was 7.2% (51/709) overall. Sensitivity, specificity, positive and negative predictive values of the io®CT assay were, respectively, 96.1% (95% Confidence Interval (CI): 86.5-99.5), 97.7% (95%CI: 96.3-98.7), 76.6% (95%CI: 64.3-86.2) and 99.7% (95%CI: 98.9-100). There was no significant difference in performance measures between fresh and frozen samples, or between symptomatic and asymptomatic participants (p>0.05). The only risk factor associated with CT infection was being a sexual contact of an individual with CT.ConclusionsThe io®CT-assay is the only 30-minute, fully automated, high-performing NAAT currently CE-marked for CT diagnosis in women, making it a highly promising diagnostic to enable specific treatment, initiation of partner notification and appropriately intensive health promotion at the point of care. Future research is required to evaluate acceptability by clinicians and patients in GUM/RSH clinics, impact on clinical pathways and patient management, and cost-effectiveness.

scholarly journals Comparative Evaluation of Initial and New Versions of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Direct Detection of Mycobacterium tuberculosis in Respiratory and Nonrespiratory Specimens

1998 ◽  
Vol 36 (3) ◽  
pp. 684-689 ◽  
Author(s):  
Fredy Gamboa ◽  
Gregorio Fernandez ◽  
Eduardo Padilla ◽  
José M. Manterola ◽  
Joan Lonca ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Direct Detection ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Direct Test ◽  
Predictive Values ◽  
Staining Methods ◽  
Sensitivity Specificity ◽  
Respiratory Specimens

We evaluated the initial version of the Amplified Mycobacterium Tuberculosis Direct Test (Gen-Probe) (AMTDT 1) and the new version of AMTDT (AMTDT 2) for the detection of Mycobacterium tuberculosis directly from respiratory and nonrespiratory samples and compared the results with those of culture and staining methods. The assays were applied to 410 respiratory and 272 nonrespiratory samples collected from 515 patients. The combination of the culture results and clinical diagnosis was considered to be the “gold standard.” Ninety-five respiratory specimens were collected from 67 patients with a diagnosis of pulmonary tuberculosis (TB) and 68 nonrespiratory specimens were collected from 61 patients with a diagnosis of extrapulmonary TB. With respiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 96%, respectively, for AMTDT 1 and 94.7, 100, 100, and 98.4%, respectively, for AMTDT 2. With nonrespiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 94%, respectively, for AMTDT 1 and 86.8, 100, 100, and 98.4%, respectively, for AMTDT 2. The overall results of AMTDT 1 and AMTDT 2 were concordant for 97% (661 of 682) of the samples. Statistically significant differences in sensitivities were found between AMTDT 1 and AMTDT 2 with respiratory specimens. It was concluded that although both nucleic acid amplification methods are rapid, sensitive, and specific for the detection of M. tuberculosis complex in all types of clinical samples, AMTDT 2 appeared to be more sensitive than AMTDT 1 when applied to smear-negative specimens. In contrast AMTDT 2 is more susceptible than AMTDT 1 to inhibitory substances in the amplification reaction. The turnaround time of AMTDT 2 is shorter (3.5 h) than that for AMTDT 1 (5 h).

scholarly journals Ultrastaging Using Ex Vivo Sentinel Lymph Node Mapping and One-Step Nucleic Acid Amplification (OSNA) in Gastric Cancer: Experiences of a European Center

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2683
Author(s):  
Bruno Märkl ◽  
Bianca Grosser ◽  
Kerstin Bauer ◽  
Dmytro Vlasenko ◽  
Gerhard Schenkirsch ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Detection Rate ◽  
Ex Vivo ◽  
Sentinel Lymph Node Mapping ◽  
Nucleic Acid Amplification ◽  
Lymph Node Mapping ◽  
Single Positive ◽  
Conventional Histology ◽  
One Step ◽  
Sensitivity Specificity

Background: In this study, the effectiveness of One-step nucleic acid amplification (OSNA) in combination with ex vivo SLN mapping is compared with conventional histology including immunohistochemistry. Methods: LNs were retrieved from gastrectomy specimens in an unfixed state. After ex vivo SLN mapping using methylene-blue, LNs were sliced to provide samples for histology and OSNA. Results: In total, 334 LNs were retrieved in the fresh state from 41 patients. SLN detection was intended in 40 cases but was successful in only 29, with a correct LN status prediction in 23 cases (79%). Excluding one case out of 41 with a failure likely caused by a processing error, OSNA showed a high effectiveness with sensitivity, specificity, and accuracy rates of 85.4%, 93.5%, and 92.4%, respectively. The LN status could be predicted in all but one case, in which the single positive LN was not eligible for OSNA testing. Moreover, OSNA evaluation led to upstaging from N0 to N+ in three cases (14%). Conclusion: The ex vivo SLN protocol used resulted in a relatively poor detection rate. However, the OSNA method was not hampered by this detection rate and proved its potential to increase the sensitivity of metastases detection.

scholarly journals Limited-Resource Preparable Chitosan Magnetic Particles for Extracting Amplification-Ready Zeptomole-Range Nucleic Acid from Complex Biofluid

2021 ◽  
Author(s):  
Sayantan Tripathy ◽  
Arunansu Talukdar ◽  
Goutam Pramanik ◽  
P. V. Rajesh ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Magnetic Particles ◽  
Limited Resource ◽  
Nucleic Acid Amplification ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction ◽  
Spin Column

<b>Layman Summary: </b>Nucleic acid extraction is a key prerequisite for any nucleic acid amplification test (NAAT) or isothermal NAAT (iNAAT) based molecular diagnosis assays.<b> </b>Existing methods utilizes spin column system for nucleic acid extraction which are unsuitable for limited resource settings. Our work explores two methods for chitosan coated magnetic particle preparation that can be executed within 6 h from commonly available chemicals with nothing but a magnetic stirrer and water bath and doable by a minimally trained person. We will also investigated the compatibility of the extracted nucleic acid with downstream NAATs such as real time LAMP, colorimetric LAMP, and real time PCR. In the process, we established the analytical sensitivity of the overall method.<div><br><div><b>Characterization methods</b>: SEM, XRD, EDX, FT-IR</div><div><br></div><div><b>Bioanalytical methods:</b> Real time LAMP, Colorimetric LAMP, Real time PCR</div></div>

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