Dataset Information

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

ABSTRACT: 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%.

SUBMITTER: Mahendra C

PROVIDER: S-EPMC8416441 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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

Mahendra Caroline C Kaisar Maria Mardalena Martini MMM Vasandani Suraj Rajan SR Surja Sem Samuel SS Tjoa Enty E Chriestya Febie F Junusmin Kathleen Irena KI Widowati Tria Asri TA Irwanto Astrid A Ali Soegianto S

Frontiers in cellular and infection microbiology 20210818

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 d ...[more]

PMID: 34485174

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Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused a global pandemic beginning in 2020, can be detected by reverse-transcription polymerase chain reaction (RT-PCR). However, owing to the urgent need for a large number of detection kits, the time spent researching and developing these kits has been shortened during the pandemic, and the kits that are being used commercially have not undergone full and independent evaluation. To ensure the accuracy of SARS-CoV-2 test results, performance verification of commercial Real-Time quantitative PCR (RT-qPCR) kits is required.MethodsThe performance of five commercial RT-qPCR diagnostic kits for SARS-CoV-2 used in China was evaluated using a coronavirus disease 2019 (COVID-19) RNA liquid performance verification reference product-manufactured by Guangzhou Bondson (BDS) Biotechnology Co., Ltd.,Guangzhou, China-that uses droplet digital RT-PCR technology combined with fluorescence quantitative PCR. The five kits of Novel Coronavirus 2019-nCoV nucleic acid detection kit (RT-qPCR method) evaluated were Da An (Da An Gene Co., Ltd. of Sun Yat-sen University), Liferiver (Shanghai ZJ Bio-Tech Co., Ltd.), Kinghawk (Beijing Kinghawk Pharmaceutical Co., Ltd.), eDiagnosis (Wuhan Easy Diagnosis Biomedicine Co., Ltd.), and Maccura (Maccura Biotechnology Co., Ltd.). Performance verification criteria included the coincidence rate, limit of detection (LoD), cross-reactivity, precision, and anti-interference. Finally, through the BDS performance verification reference product kit, clinical samples are used to verify its clinical diagnostic efficacy.ResultsThe coincidence rate was 100% for all kits except for Kinghawk, which was 95%. The LoD for Da An, eDiagnosis and Maccura was 250copies/mL, and it was 1000 copies/ml for Liferiver. Kinghawk was not able to detect its advertised LoD of 500 copies/ml. The cross-reactivity test results were all negative. Moreover, all kits had a coefficient of variation less than 5%; however, Liferiver showed the best precision. Da An, Liferiver, and eDiagnosis showed higher sensitivity to the nucleocapsid (N) gene than they did to the open reading frame (ORF) 1ab genes. Anti-interference results for all five kits were positive. The results of clinical diagnostic efficacy were that the specificity of the four kits was 1.000 (0.877-1.000), the sensitivity of Da An was 1.000 (0.850-1.000), Liferiver was 0.964 (0.798-0.998), Maccura was 0.893 (0.706-0.972), and eDiagnosis was 0.857 (0.664-0.953).ConclusionsAll commercial RT-qPCR diagnostic kits for SARS-CoV-2 passed the BDS performance verification, except for Kinghawk (batch No:20200608113) which failed to detect the LoD of 500 copies/mL. Da An and Liferiver have excellent clinical diagnostic specificity and sensitivity. This study can provide guidance for the selection or optimization of RT-qPCR diagnostic test kits for SARS-CoV-2.

Project description:Since December 2019, SARS-CoV-2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS-CoV-2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre-analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS-CoV-2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost-effective multiplex reverse-transcription real-time PCR assay that can be used to detect SARS-CoV-2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS-CoV-2 by RT-qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.

Dataset Information

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

Publications

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

Similar Datasets

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