scholarly journals Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 239 ◽  
Author(s):  
Jen-Hui Tsou ◽  
Hongjie Liu ◽  
Sanford A. Stass ◽  
Feng Jiang
Keyword(s):  
Uv Light ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Growth Factor Receptor ◽  
Rapid Test ◽  
Diagnostic Value ◽  
Sensitive Detection ◽  
Nasopharyngeal Swab ◽  
M Gene ◽  
Single Temperature

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for controlling the pandemic of coronavirus disease 2019. Polymerase chain reaction (PCR)-based technique is the standard test for detection of SARS-CoV-2, which, however, requires complicated sample manipulation (e.g., RNA extraction) and is time-consuming. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could precisely detect Human papillomavirus and somatic mutations of Epidermal growth factor receptor gene and Kirsten rat sarcoma viral oncogene homolog gene in plasma. The objective of this study was to develop CRISPR as a rapid test for sensitive detection of SARS-CoV-2. We first combined reverse transcription-isothermal recombinase polymerase amplification and CRSIPR to detect SARS-CoV-2 in genomic RNA of cells infected with the virus. The CRISPR assay with guide RNA against the M gene of SARS-CoV-2 had a sensitivity of 0.1 copies per µL for detection of the virus. We then used the CRSIPR assay to directly analyze raw SARS-CoV-2 samples. The CRISPR assay could sensitively detect SARS-CoV-2 in one hour without RNA extraction. This assay can be performed at a single temperature and with minimal equipment. The results were immediately visualized either by a UV light illuminator or paper strips. The diagnostic value of the test was confirmed in nasopharyngeal swab specimens. Altogether, we have developed a rapid CRISPR test for sensitive detection of SARS-CoV-2.

DETEKSI ANTIBODI MULTIPEL HEPATITIS C DALAM DARAH DONOR

2016 ◽  
Vol 21 (3) ◽  
pp. 261
Author(s):  
Ranti Permatasari ◽  
Aryati Aryati ◽  
Budi Arifah
Keyword(s):  
Hepatitis C ◽  
Predictive Value ◽  
Core Protein ◽  
Rapid Test ◽  
Antibody Test ◽  
Diagnostic Value ◽  
Hcv Infection ◽  
Antibody Detection ◽  
Hcv Rna ◽  
Hcv Antibody

Hepatitis C (HCV) infection could be spread by blood transfusion. Screening of HCV in donor blood could prevent HCV infection to the recipient. HCV antibody test using rapid test of multiple antibody detection by immunochromatography method is an easy and rapid test that could detect four HCV antibodies separately. The aim of this study was to evaluate the diagnostic value of antibody HCV using multiple antibody detection rapid test in diagnosing HCV infection. This was an analytical observational study with a cross sectional design. The samples consisted of 42 donors’ blood serum from the Surabaya Branch of the Indonesian Red Cross which underwent HCV infection test using ELISA method. The samples were then tested using PCR HCV RNA as the gold standard and antibody HCV multiple antibodydetection rapid test The diagnostic value of HCV antibody test using multiple antibody detection rapid test by immunochromatography method showed a diagnostic sensitivity of 100%, diagnostic specificity of 75%, positive predictive value of 66.7% and negative predictive value of 100%, a diagnostic efficiency of 83.3%, with a positive probability ratio of 4 times. The most often positive antibody pattern was four (4) positive antibodies (core protein, NS3, NS4 and NS5). Core protein (CP) and NS3 were the most often positive antibodies. Based on this study result, the HCV antibody test using multiple antibody detection rapid test by immunochromatography method has a good diagnostic value.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals SARS-CoV-2 in peritoneal swabs from asymptomatic patients undergoing emergency abdominal surgery

2021 ◽  
Vol 2021 (4) ◽  
Author(s):  
Jasim AlAradi ◽  
Rawan A Rahman AlHarmi ◽  
Mariam AlKooheji ◽  
Sayed Ali Almahari ◽  
Mohamed Abdulla Isa ◽  
...  
Keyword(s):  
Peritoneal Fluid ◽  
Case Series ◽  
Rapid Test ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Protective Measures ◽  
The Public ◽  
Asymptomatic Patients ◽  
Number Of Patients ◽  
Viral Transfer

Abstract This is a case series of five patients with acute abdomen requiring surgery who tested positive for coronavirus disease 2019 (COVID-19) and were asymptomatic, with the purpose of detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in peritoneal fluid. Nasopharyngeal swab was done as a prerequisite for admission or prior to admission as part of random testing. Two methods of viral testing were employed: Xpert® Xpress SARS-CoV-2 (rapid test) and real-time reverse transcription polymerase chain reaction (RT-PCR). Either or both tests were done, with the former performed for patients requiring surgery immediately. Surgery was performed within 24–36 h from admission. Peritoneal fluid swabs were obtained for the detection of SARS-CoV-2 using RT-PCR test. Swabs were immediately placed in viral transfer media and delivered to the public health laboratory in an ice bag. SARS-CoV-2 was not detected in peritoneal swabs. Due to the limited number of patients, further studies are required; yet, protective measures should still be taken by surgeons when dealing with COVID-19 cases.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Arginase 1 (Arg1) as an Up-Regulated Gene in COVID-19 Patients: A Promising Marker in COVID-19 Immunopathy

2021 ◽  
Vol 10 (5) ◽  
pp. 1051 ◽  
Author(s):  
Afshin Derakhshani ◽  
Nima Hemmat ◽  
Zahra Asadzadeh ◽  
Moslem Ghaseminia ◽  
Mahdi Abdoli Shadbad ◽  
...  
Keyword(s):  
Immune Responses ◽  
Whole Blood ◽  
Roc Analysis ◽  
Operating Characteristic ◽  
Rna Extraction ◽  
Diagnostic Value ◽  
Healthy Individuals ◽  
Cdna Synthesis ◽  
Arginase 1 ◽  
Global Pandemic

Background: The coronavirus disease 2019 (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic. It is well-established that SARS-CoV-2 infection can lead to dysregulated immune responses. Arginase-1 (Arg1), which has a pivotal role in immune cells, can be expressed in most of the myeloid cells, e.g., neutrophils and macrophages. Arg1 has been associated with the suppression of antiviral immune responses. Methods: Whole blood was taken from 21 COVID-19 patients and 21 healthy individuals, and after RNA extraction and complementary DNA (cDNA) synthesis, gene expression of Arg1 was measured by real-time PCR. Results: The qPCR results showed that the expression of Arg1 was significantly increased in COVID-19 patients compared to healthy individuals (p < 0.01). The relative expression analysis demonstrated there were approximately 2.3 times increased Arg1 expression in the whole blood of COVID-19 patients. Furthermore, the receiver operating characteristic (ROC) analysis showed a considerable diagnostic value for Arg1 expression in COVID-19 (p = 0.0002 and AUC = 0.8401). Conclusion: Arg1 might be a promising marker in the pathogenesis of the disease, and it could be a valuable diagnostic tool.

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