Performance Evaluation of Developed Bangasure™ Multiplex rRT-PCR Assay for SARS-CoV-2 Detection in Bangladesh: A Blinded Observational Study at Two Different Sites

Abstract

In this study, we evaluated the performance of the in-house developed rRT-PCR assay for SARS-CoV-2 RNA targeting the envelope (E) and nucleocapsid (N) genes with internal control as human RNase P. A total of 50 positive samples and 50 negative samples of SARS-CoV-2 were tested by a reference kit at site 1 and a subset (30 positives and 16 negatives) of these samples are tested blindly at site 2. The limit of detection (LoD) was calculated by using a replication-deficient complete SARS-CoV-2 genome and known copy numbers, where Pseudo-virus samples were used to evaluate accuracy. On site 1, among the 50 SARS-CoV-2 positive samples 24, 18, and eight samples showed high (Ct < 26), moderate (26 < Ct ≤ 32), and low (32 < Ct ≤ 38) viral load, respectively, whereas in site 2, out of 30 SARS-CoV-2 positive samples, high, moderate, and low viral loads were found in each of the 10 samples. However, SARS-CoV-2 was not detected in the negative sample. So, in-house assays at both sites showed 100% sensitivity and specificity with no difference observed between RT PCR machines. The Ct values of the in-house kit had a very good correlation with the reference kits. LoD was determined as 100 copies/mL. It also displayed 100% accuracy in mutant and wild-type SARS-CoV-2 virus. This Bangasure™ RT-PCR kit shows excellent performance in detecting SARS-CoV-2 viral RNA compared to commercially imported CE-IVD marked FDA authorized kits.

Keywords: Bangasure™; LoD; Nucleocapsid; SARS-CoV-2; rRT-PCR.

Figure 1

Amplification curves for each of the target genes in singleplex: (A) E gene, (B) N gene, (C) RNase P gene and (D) Multiplex assay with the optimized combination of primer-probes.

Figure 2

Calculation of calibration curves through singleplex and multiplex rRT-PCR for different copies of positive controls of E, N2 and RNase P. (A) Standard curve for E, (B) Standard curve for N and (C) Standard curve for RNase P.

Figure 2

Calculation of calibration curves through singleplex and multiplex rRT-PCR for different copies of positive controls of E, N2 and RNase P. (A) Standard curve for E, (B) Standard curve for N and (C) Standard curve for RNase P.

Figure 3

Detection of the SARS-CoV-2 variants of concerns by Bangasure^TM multiplex rRT-PCR kit. (A) Wuhan variant (wild type), (B) UK Variant (B.1.1.7), (C) South African Variant (B.1.351), (D) Brazilian variant (P.1) and (E) Omicron Variant (B.1.1.529) from clinical specimen (OM574617).

Figure 3

Detection of the SARS-CoV-2 variants of concerns by Bangasure^TM multiplex rRT-PCR kit. (A) Wuhan variant (wild type), (B) UK Variant (B.1.1.7), (C) South African Variant (B.1.351), (D) Brazilian variant (P.1) and (E) Omicron Variant (B.1.1.529) from clinical specimen (OM574617).

Figure 4

Pearson correlation analysis of Ct values of E, N, RNase P between in house kit and reference kits. (A) Pearson correlation between N, RNase P, E gene between In house and Sansure kit at site 1 (Sansure has ORF1ab gene instead of E gene), (B) Pearson correlation between N, RNase P, E gene between In house and One copy kit at site 1, (C) Pearson correlation between N, RNase P, E gene between In house and Sansure kit at site 1 (Sansure has ORF1ab gene instead of E gene).

Figure 5

Ct values of individual genes included in in-house kit on two different RT machines used at two sites.