Query: Nucleic acid test isothermal amplification SARS-COV-2

title: Comparison of a laboratory developed test targeting the envelope gene with three nucleic acid amplification tests for detection of SARS CoV 2
abstract: Numerous nucleic acid amplification tests, including real time, reverse transcription PCR (rRT PCR) and isothermal amplification methods, have been developed to detect SARS CoV 2 RNA, including many that have received emergency use authorization (EUA) There is a need to assess their test performance relative to one another.  The aim of this study was to compare the test performance of a high complexity laboratory developed rRT PCR EUA from Stanford Health Care (SHC) targeting the SARS CoV 2 envelope (E) gene with other tests: the Atila isothermal amplification assay targeting the nucleocapsid (N) gene and open reading frame 1ab (ORF1ab) the Altona E and spike (S) multiplex, real time RT PCR, and the US Centers for Disease Control and Prevention (CDC) N1 and N2 rRT PCRs.  A diagnostic comparison study was performed by testing nasopharyngeal samples from persons under investigation for coronavirus disease 2019  Assay performance was assessed by percent agreement and Cohen's kappa coefficient.  Positive percent agreement with the SHC EUA reference assay was 82.8% (95% confidence interval (CI) 65.0 to 92.9) for Atila, 86.7% (95% CI 69.7 to 95.3) for the Altona E and S targets, and 86.7% (95% CI 69.7 to 95.3) and 90.0% (95% CI 73.6 to 97.3) for the CDC N1 and N2 targets, respectively. All assays demonstrated 100% negative percent agreement. Kappa coefficients ranged from 0.86 to 0.92, indicating excellent agreement.  Performance was comparable among the SARS CoV 2 nucleic acid amplification methods tested, with a limited number of discrepancies observed in specimens with low viral loads.

title: Multiple centre clinical evaluation of an ultrafast single tube assay for SARS CoV 2 RNA
abstract: To evaluate the performance of an ultra fast single tube nucleic acid isothermal amplification detection assay for SARS CoV 2 RNA using clinical samples from multiple centers.  A reverse transcription recombinase aided amplification (RT RAA) assay for SARS CoV 2 was conducted within 15minutesat39degC with portable instruments after addition of extracted RNA. The clinical performance of RT RAA assay was evaluated using 947 clinical samples from five institutions in four regions of China, and the approved commercial real time fluorescent RT PCR (qRT PCR) kits were used for parallel detection. The sensitivity and specificity of RT RAA were compared and analyzed.  The RT RAA test results of 926 samples were consistent with those of qRT PCR (330 were positive, 596 were negative) and 21 were inconsistent. The sensitivity and specificity of RT RAA was 97.63% [330/338, 95% confidence interval (CI) 95.21 to 98.90] and 97.87% (596/609, 95% CI: 96.28 to 98.81) respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 96.21% (330/343, 95% CI: 93.45 to 97.88) and 98.68% (596/604, 95% CI: 97.30 to 99.38) respectively. The total coincidence rate was 97.78% (926/947, 95% CI: 96.80 to 98.70) and the Kappa was 0.952 (P <0.05)  With comparable sensitivity and specificity to the commercial qRT PCR kits, RT RAA assay for SARS CoV 2 exhibited distinctive advantages of simplicity and rapidity in terms of operation and turn around time.

title: Direct diagnostic testing of SARS CoV 2 without the need for prior RNA extraction.
abstract: The COVID 19 pandemic has resulted in an urgent global need for rapid, point of care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low cost, scalable, point of care testing. We report the development of a highly sensitive and robust, easy to implement, SARS CoV 2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/l, and can be run using a simple heat block.

title: An enhanced isothermal amplification assay for viral detection
abstract: Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS CoV 2, FIND (Fast Isothermal Nucleic acid Detection) based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available. One sentence  Sensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS CoV 2 from patient samples.

title: Recent advances and perspectives of nucleic acid detection for coronavirus
abstract: The recent pneumonia outbreak caused by a novel coronavirus (SARS CoV 2) in Chinais posing a great threat to global public health. Therefore, rapid and a accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments, saving people's lives and preventing epidemics. It is important to establish a quick standard diagnostic test for the detection of the infectious disease (COVID 19) to prevent subsequent secondary spread. Polymerase chain reaction (PCR) is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity. Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler operation. A variety of improved or new approaches also have been developed. This review summarizes the currently available detection methods for coronavirus nucleic acid. It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coronavirus infection.

title: A comparative study of isothermal nucleic acid amplification methods for SARS CoV 2 detection at point of care
abstract: COVID 19, caused by the novel coronavirus SARS CoV 2, has spread worldwide and put most of the world under lockdown. Despite that there have been emergently approved vaccines for SARS CoV 2, COVID 19 cases, hospitalizations, and deaths have remained rising. Thus, rapid diagnosis and necessary public health measures are still key parts to contain the pandemic. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS CoV 2 detection based on loop mediated isothermal amplification (LAMP) cross priming amplification (CPA) and polymerase spiral reaction (PSR) were designed and evaluated. The three methods showed the same limit of detection (LOD) value of 1 copy of the targeted gene per reaction. However, for the direct detection of SARS CoV 2 genomic RNA, LAMP outperformed both CPA and PSR, exhibiting the LOD value of roughly 43.14 genome copies/reaction. The results can be read with the naked eye within 45 minutes, without cross reactivity to closely related coronaviruses. Moreover, the direct detection of SARS CoV 2 RNA in simulated patient specimens by iNAATs was also successful. Finally, the ready to use lyophilized reagents for LAMP reactions were shown to maintain the sensitivity and LOD value of the liquid assays. The results indicate that the colorimetric lyophilized LAMP kit developed herein is highly suitable for detecting SARS CoV 2 nucleic acids at point of care.

title: Analysis of adjunctive serological detection to nucleic acid test for severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) infection diagnosis
abstract: The severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) caused coronavirus disease 2019 (COVID 19) epidemic in China, December 2019. The clinical features and treatment of COVID 19 patients remain largely elusive. However, accurate detection is required for SARS CoV 2 infection diagnosis. We aimed to evaluate the antibodies based test and nucleic acid based test for SARS CoV 2 infected patients.  We retrospectively studied 133 patients diagnosed with SARS CoV 2 and admitted to Renmin Hospital of Wuhan University, China, from January 23 to March 1, 2020. Demographic data, clinical records, laboratory tests, and outcomes were collected. Data were accessed by SARS CoV 2 IgM IgG antibody test and real time reverse transcriptase PCR (RT PCR) detection for SARS CoV 2 nucleic acid in COVID 19 patients.  Of 133 COVID 19 patients, there were 44 moderate cases, 52 severe cases, and 37 critical cases with no differences in gender and age among three subgroups. In RT PCR detection, the positive rate was 65.9% 71.2% and 67.6% in moderate, severe, and critical cases, respectively. Whereas the positive rate of IgM/IgG antibody detection in patients was 79.5%/93.2% 82.7%/100% and 73.0%/97.3% in moderate, severe, and critical cases, respectively. Moreover, the IgM and IgG antibodies concentrations were also examined with no differences among three subgroups.  The IgM IgG antibody test exhibited a useful adjunct to RT PCR detection, and improved the accuracy in COVID 19 diagnosis regardless of the severity of illness, which provides an effective complement to the false negative results from a nucleic acid test for SARS CoV 2 infection diagnosis after onsets.

title: Rapid direct nucleic acid amplification test without RNA extraction for SARS CoV 2 using a portable PCR thermocycler
abstract: There is an ongoing worldwide coronavirus disease 2019 (Covid 19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT PCR) typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction free PCR (DIRECT PCR) detection of SARS CoV 2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one step inhibitor resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single tube homogeneous reaction within 36 minutes. This minimized hands on time, reduces turnaround time for sample to result and obviates the need for RNA purification reagents. It could enable wider use of Covid 19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.

title: High surety isothermal amplification and detection of SARS CoV 2, including with crude enzymes
abstract: Isothermal nucleic acid amplification tests (iNAT) such as loop mediated isothermal amplification (LAMP) are good alternatives to polymerase chain reaction (PCR) based amplification assays, especially for point of care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non sequence specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS CoV 2 LAMP assays that relied on non sequence specific readout into assays that can be visually read using sequence specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one pot operation of both individual and multiplex LAMP OSD assays and demonstrate detection of SARS CoV 2 virions in crude human saliva.

title: Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV 2) by reverse transcription loop mediated isothermal amplification
abstract: Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV 2 or 2019 nCoV) and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID 19) is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT PCR) is currently the standard for SARS CoV 2 detection; however, Reverse Transcription Loop Mediated Isothermal Amplification (RT LAMP) may allow for faster and cheaper field based testing at point of risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30 45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS CoV 2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID 19 patients was also tested. The samples were tested using RT LAMP as well as by conventional qRT PCR. Specificity of the RT LAMP was evaluated by also testing against other related coronaviruses. RT LAMP specifically detected SARS CoV 2 in both simulated patient samples and clinical specimens. This test was performed in 30 45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.