scholarly journals Wide application of minimally processed saliva on multiple RT-PCR kits for SARS-CoV-2 detection in Indonesia

2021 ◽  
Author(s):  
Caroline Mahendra ◽  
Maria M. M. Kaisar ◽  
Suraj R. Vasandani ◽  
Sem Samuel Surja ◽  
Enty Tjoa ◽  
...  
Keyword(s):  
Target Genes ◽  
Local Population ◽  
Rna Extraction ◽  
Human Saliva ◽  
Attractive Alternative ◽  
Rt Pcr ◽  
Sampling Device ◽  
Agreement Rate ◽  
Extraction Step ◽  
Oropharyngeal Swab

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, it is recommended to evaluate a proposed workflow amongst the local population. Here, we aim to validate collection and treatment of human saliva as a direct specimen for RT-qPCR based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimen and remained stable for five days refrigerated or room temperature storage. The method of processing saliva specimen described in this report is free from RNA-extraction step, thereby reduces cost, time, and manpower required for processing samples. The developed method was validated for use on three COVID-19 RT-PCR kits commercially available. Our developed method achieved 85% agreement rate when compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a specimen sampling device, QuickSpit(TM), collection was found to be more convenient for individuals and improved agreement rate to 90%.

scholarly journals Wide Application of Minimally Processed Saliva on Multiple RT-qPCR Kits for SARS-CoV-2 Detection in Indonesia

2021 ◽  
Vol 11 ◽  
Author(s):  
Caroline Mahendra ◽  
Maria Mardalena Martini Kaisar ◽  
Suraj Rajan Vasandani ◽  
Sem Samuel Surja ◽  
Enty Tjoa ◽  
...  
Keyword(s):  
Target Genes ◽  
Local Population ◽  
Rna Extraction ◽  
Extraction Process ◽  
Human Saliva ◽  
Attractive Alternative ◽  
Sampling Device ◽  
Agreement Rate ◽  
Commercial Kits ◽  
The Cost

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, evaluating a proposed workflow amongst the local population is recommended. Here, we aim to validate the collection and treatment of human saliva as a direct specimen for RT-qPCR-based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimens and remained stable for five days either refrigerated or stored at room temperature. The method of processing saliva specimens described in this report bypasses the need for an RNA-extraction process, thereby reducing the cost, time, and manpower required for processing samples. The developed method was tested across nine commercial kits, including the benchmark, to demonstrate its wide applicability on multiple existing workflows. Our developed method achieved an 86% overall agreement rate compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a saliva sampling device, the collection was found to be more convenient for individuals and improved the overall agreement rate to 97%.

scholarly journals A Rapid SARS-CoV-2 RT-PCR Assay for Low Resource Settings

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 739
Author(s):  
Arunkumar Arumugam ◽  
Matthew L. Faron ◽  
Peter Yu ◽  
Cole Markham ◽  
Michelle Wu ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Qpcr Assay ◽  
Water Bath ◽  
Raspberry Pi ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Low Resource Settings ◽  
Clinical Sensitivity ◽  
Low Resource ◽  
Extraction Step

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

scholarly journals An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1370-1374 ◽  
Author(s):  
Mohamad Chikh-Ali ◽  
Stewart M. Gray ◽  
Alexander V. Karasev
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Rna Extraction ◽  
Leaf Tissue ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Potato Leaf ◽  
Extraction Step ◽  
First Time ◽  
Virus Testing

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

Development of a TaqMan® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses

2005 ◽  
Vol 124 (1-2) ◽  
pp. 65-71 ◽  
Author(s):  
Boris Pastorino ◽  
Maël Bessaud ◽  
Marc Grandadam ◽  
Severine Murri ◽  
Hugues J. Tolou ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Extraction Step ◽  
Detection And Quantification

scholarly journals Sensitivity of nasopharyngeal, oropharyngeal and nasal washes specimens for SARS-CoV-2 detection in the setting of sampling device shortage

2020 ◽  
Author(s):  
Adrien Calame ◽  
Lena Mazza ◽  
Adriana Renzoni ◽  
Laurent Kaiser ◽  
Manuel Schibler
Keyword(s):  
Culture Medium ◽  
Alternative Methods ◽  
Analytical Sensitivity ◽  
Sample Collection ◽  
Rt Pcr ◽  
Sampling Device ◽  
Viral Transport ◽  
Viral Transport Medium ◽  
A Cell ◽  
Oropharyngeal Swab

In the context of an unprecedented shortage of nasopharyngeal swabs (NPS) or sample transport media during the coronavirus disease 2019 (COVID-19) crisis, alternative methods for sample collection are needed. To address this need, we validated a cell culture medium as a viral transport medium, and compared the analytical sensitivity of SARS-CoV-2 real-time RT-PCR in nasal wash (NW), oropharyngeal swab (OPS) and NPS specimens. Both the clinical and analytical sensitivity were comparable in these three sample types. OPS and NW specimens may therefore represent suitable alternatives to NPS for SARS-CoV-2 detection.

scholarly journals SARS-CoV-2 direct real-time polymerase chain reaction testing in laboratories with shortage challenges

2021 ◽  
Vol 16 (2) ◽  
pp. 133-139
Author(s):  
Taghreed Alaifan ◽  
Asmaa Altamimi ◽  
Dalia Obeid ◽  
Turki Alshehri ◽  
Shaihana Almatrrouk ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rna Extraction ◽  
Threshold Value ◽  
Limited Resources ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Viral Transport ◽  
Extraction Step ◽  
Polymerase Chain

Aim: In our study, we propose the use of direct real-time polymerase chain reaction (RT-PCR), this test does not require extraction or a preheating step, which saves a lot of cost, labor, processing time and provides a solution for supply shortage. Materials & methods: We assayed 185 nasopharyngeal samples stored in viral transport media. The indirect method was done with RNA extraction step, and the direct RT-PCR was done without an extraction step, both assays were evaluated on a commercially validated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kit targeting E gene. Results: Our assay showed a sensitivity of 79%, a specificity of 100% and the agreement between methods was 72%. Conclusion: Overall, this simple direct RT-PCR approach can be utilized as a qualitative diagnostic tool for emergency SARS-CoV-2 surveillance in countries with limited resources and may help laboratories to continue testing and at greater frequency despite supply shortages, although with delay in cycle threshold value in comparison with indirect RT-PCR.

scholarly journals EasyCOV : LAMP based rapid detection of SARS-CoV-2 in saliva

2020 ◽  
Author(s):  
Nicolas L’Helgouach ◽  
Pierre Champigneux ◽  
Francisco Santos Schneider ◽  
Laurence Molina ◽  
Julien Espeut ◽  
...  
Keyword(s):  
Healthcare Worker ◽  
Rna Extraction ◽  
Virus Detection ◽  
Rt Pcr ◽  
Double Blind ◽  
Detection Assay ◽  
Extraction Step ◽  
Late Control ◽  
Enhance Efficiency ◽  
Clinical Conditions

AbstractCovid-19 crisis showed us that rapid massive virus detection campaign is a key element in SARS-CoV-2 pandemic response. The classical RT-PCR laboratory platforms must be complemented with rapid and simplified technologies to enhance efficiency of large testing strategies.To this aim, we developed EasyCOV, a direct saliva RT-LAMP based SARS-CoV-2 virus detection assay that do not requires any RNA extraction step. It allows robust and rapid response under safe and easy conditions for healthcare workers and patients.EasyCOV test was assessed under double blind clinical conditions (93 asymptomatic healthcare worker volonteers, 10 actively infected patients, 20 former infected patients tested during late control visit). EasyCOV results were compared with classical laboratory RT-PCR performed on nasopharyngeal samples.Our results show that compared with nasopharyngeal laboratory RT-PCR, EasyCOV SARS-CoV-2 detection test has a sensitivity of 72.7%. Measured on healthcare worker population the specificity was 95.7%. LAMP technology on saliva is clearly able to identify subjects with infectivity profile. Among healthcare worker population EasyCOV test detected one presymptomatic subject.Because it is simple, rapid and painless for patients, EasyCOV saliva SARS-Cov-2 detection test may be useful for large screening of general population.

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.

Osteopontin expressed by renal tubular epithelium mediates interstitial monocyte infiltration in rats

2000 ◽  
Vol 278 (1) ◽  
pp. F110-F121 ◽  
Author(s):  
Hirokazu Okada ◽  
Kenshi Moriwaki ◽  
Raghuram Kalluri ◽  
Tsuneo Takenaka ◽  
Hiroe Imai ◽  
...  
Keyword(s):  
Target Genes ◽  
Renal Plasma Flow ◽  
Mrna Level ◽  
Antisense Oligodeoxynucleotide ◽  
Tubular Epithelium ◽  
Rt Pcr ◽  
Goodpasture Syndrome ◽  
Renal Tubular Epithelium ◽  
Protein Excretion ◽  
Renal Tubular

In this study, we have shown that intravenously administered antisense oligodeoxynucleotide (ODN) was demonstrated to be taken up by tubular epithelium, after which it blocked mRNA expression of target genes in normal and nephritic rats. Therefore, we injected osteopontin (OPN) antisense ODN to Goodpasture syndrome (GPS) rats every second day between days 27 and 35, the time when renal OPN expression increased and interstitial monocyte infiltration was aggravated. In parallel to blockade of tubular OPN expression, this treatment significantly attenuated monocyte infiltration and preserved renal plasma flow in GPS rats at day 37, compared with sense ODN-treated and untreated GPS rats. No significant changes were observed in OPN mRNA level by RT-PCR and histopathology of the glomeruli after ODN treatment, which was compatible with an absence of differences in the urinary protein excretion rate. In conclusion, OPN expressed by tubular epithelium played a pivotal role in mediating peritubular monocyte infiltration consequent to glomerular disease.

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.

Sign in / Sign up

Export Citation Format

Share Document