Development of an in-house primer design: Nested polymerase chain reaction for coronavirus disease 2019 detection based on open reading frame 1a/b and spike

Abstract

Background and Aim: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 1 million people and caused more than 100,000 deaths in Indonesia. This condition was augmented by a less advanced health system, especially in providing diagnostic facilities for the novel coronavirus, and the high mutation rate of the novel coronavirus, which may promote the generation of specific strains in Indonesia. This study aimed to propose a specific primer (in-house primer) toward open reading frame 1a/b (ORF1ab) and the spike protein gene of SARS-CoV-2 to detect SARS-CoV-2 and to analyze the presence of mutations. Materials and Methods: One hundred and nine samples were collected from patients in Malang, East Java, Indonesia. The samples were extracted using QIAamp viral RNA kits. The in-house primer was designed using Clone Manager 9.0 and amplified using nested polymerase chain reaction (PCR). Then, the amplicon was analyzed through sequencing. The detection results were compared with those obtained using the quantitative PCR (qPCR). Results: Nested PCR was 74.3% positive, whereas qPCR was 45.9% positive. Furthermore, sequencing analysis of the amplicon revealed the mutation at locations T3187C, T2889C/T, G3189T (spike), and C364T (ORF1ab). The sensitivity and specificity of nested PCR were 92.6% and 43.6%, respectively. This result indicated that the in-house primer performed well at screening. Conclusion: In-house primers could detect SARS-CoV-2 and mutations in samples from Malang, East Java, Indonesia. In the future, this method could be recommended as a screening tool for monitoring SARS-CoV-2 infection.

Keywords

• nested polymerase chain reaction
• open reading frame 1a/b
• primer
• severe acute respiratory syndrome coronavirus 2
• spike