Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2

Tombuloğlu, Hüseyin; Sabit, Hussein; Al-Suhaimi, Ebtesam A.; Jindan, Reem Al; Alkharsah, Khaled R.

doi:10.1371/journal.pone.0250942

Cited by 49 publications

(45 citation statements)

References 36 publications

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“…RT-PCR tests have been developed to target RdRP , E , N , or S genes 10 , 21 , 22 . Among these, those targeting RdRP gene were found to be the ones with the highest analytical sensitivity 9 , 23 .…”

Section: Introductionmentioning

confidence: 99%

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Tombuloğlu

Sabit

Al‐Khallaf

et al. 2022

Sci Rep

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Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

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Section: Introductionmentioning

confidence: 99%

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Tombuloğlu

Sabit

Al‐Khallaf

et al. 2022

Sci Rep

Self Cite

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“…In our study, the sex distribution was almost balanced. Previous studies also showed no substantial difference between genders, 51% boys in Italy until July 21, 2020 [ 11 ], 57% in the United States [ 21 ], and 56.6% boys in China [ 9 ]. In another study on Iranian children by Soltani et al [ 19 ], 46.7% of the evaluated 30 children were male [ 19 ].…”

Section: Discussionmentioning

confidence: 95%

“…RNAseP gene was used as an internal control. All runs were performed using the RT-PCR device (Rotor gene Q, QIAGEN, Germany) according to the kit instructions [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

Demographic, clinical, and paraclinical characteristics of COVID-19 pediatric cases in southeast Iran

Soleimani¹,

Akbarirad

Shahri

et al. 2021

Antimicrob Resist Infect Control

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Background Even though children seem to be less vulnerable to the Coronavirus disease 2019 (COVID-19) infection, still a diverse range of clinical presentations and symptoms have been reported in children. Few studies assessed the clinical presentations of COVID-19 among Iranian children. We aimed to evaluate the clinical and paraclinical characteristics of COVID-19 infected children. Methods All COVID-19 suspected and confirmed children were referred to the Ali-ibn-Abitaleb Hospital, Zahedan, Iran. Patients were included in this longitudinal study. Patients were evaluated at admission and during hospitalization. Patients with some of the main COVID symptoms with positive PCR test were defined as confirmed cases. Clinical, imaging and laboratory results were collected for all patients. Results A total of 62 patients participated in this study. The male:female ratio was 1:1.03. There was a significant difference in fatigue prevalence between age groups (P = 0.002). There was no significant difference between groups in terms of fever duration (P = 0.624) and maximum temperature (P = 0.629). There was a significant difference between PCR positive and negative patients in terms of neurologic signs (P = 0.003), Intensive care unit admission (P = 0.001), white blood cell (P = 0.047). Conclusions Even though our population was small, most of the findings matched other studies conducted on pediatric cases in Iran or other countries. It was also found that some clinical features such as pneumonia, cough, diarrhea, and tachycardia at admission time were statistically different among age groups.

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“… 30 Nuclear acid sequence-based amplification (NASBA), restriction enzyme analysis (REA), direct immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and reverse transcription polymerase chain reaction (RT-PCR) are among the most frequently used methods for virus detection. 31 Multiple PCR can simultaneously amplify several nucleic acid targets in a single sample. The standard method for the detection of PCR products, agarose gel electrophoresis, is usually combined with Southern blot to determine sequence specificity.…”

Section: Current Methods For the Detection Of Sars-cov-2 And Its Mutantsmentioning

confidence: 99%

Multiplex Biosensing for Simultaneous Detection of Mutations in SARS-CoV-2

Jiang

Juhas

et al. 2021

ACS Omega

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COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has become the world’s largest public health emergency of the past few decades. Thousands of mutations were identified in the SARS-CoV-2 genome. Some mutants are more infectious and may replace the original strains. Recently, B.1.1.7(Alpha), B1.351(Beta), and B.1.617.2(Delta) strains, which appear to have increased transmissibility, were detected. These strains accounting for the high proportion of newly diagnosed cases spread rapidly over the world. Particularly, the Delta variant has been reported to account for a vast majority of the infections in several countries over the last few weeks. The application of biosensors in the detection of SARS-CoV-2 is important for the control of the COVID-19 pandemic. Due to high demand for SARS-CoV-2 genotyping, it is urgent to develop reliable and efficient systems based on integrated multiple biosensor technology for rapid detection of multiple SARS-CoV-2 mutations simultaneously. This is important not only for the detection and analysis of the current but also for future mutations. Novel biosensors combined with other technologies can be used for the reliable and effective detection of SARS-CoV-2 mutants.

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Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2

Cited by 49 publications

References 36 publications

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes

Demographic, clinical, and paraclinical characteristics of COVID-19 pediatric cases in southeast Iran

Multiplex Biosensing for Simultaneous Detection of Mutations in SARS-CoV-2

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