A Novel Point Mutation in the N Gene of SARS-CoV-2 May Affect the Detection of the Virus by Reverse Transcription-Quantitative PCR

LETTER

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, laboratory testing to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription-quantitative PCR (RT-qPCR) has played a central role in mitigating the spread of the virus (1). Soon after the viral genome sequences were available, several RT-qPCR assays were developed and made available by the World Health Organization (WHO) for public use (https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf). The primer and probe sequences for these assays were chosen from multiple target genes within the viral genome, such as the E gene, RdRp gene, ORF1ab, and N gene. Many commercial and laboratory-developed assays were developed for SARS-CoV-2 detection based on these primer and probe sequences. The large-scale sustained person-to-person transmission of SARS-CoV-2 has led to many mutational events, some of which may affect the sensitivity and specificity of available PCR assays (2). Recently, mutations in the E gene (C26340T) and N gene (C29200T) affecting the detection of target genes by two commercial assays were reported for 8 and 1 patients, respectively. Interestingly, both mutations are of the C→T type, a common single nucleotide polymorphism (SNP) that may be associated with strong host cell mRNA editing mechanisms known as apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminase (3, 4). Another study found a G→U substitution in position 29140 that affected the sensitivity of detection of N gene-based assays (5). Here, we report a novel N gene mutation (C29200A) seen in 3 patients which affected the detection of the SARS-CoV-2 N gene by a commercial assay.

Cepheid Xpert Xpress SARS-CoV-2 (Xpert) is an FDA-approved assay for COVID-19 under emergency use authorization (EUA). The Xpert assay is based on a multiplex PCR that includes both E gene and N gene targets for SARS-CoV-2 detection. The assay was implemented in our laboratory at Sidra Medicine, a pediatric referral center in Qatar, in June 2020. Since then, a total of 8,800 samples have been tested by Xpert, of which 365 (4.1%) were positive. Occasionally, discrepant results were seen (∼2.5% of all positive results) for E gene and N gene targets, which were reported as “presumptive positive” by the Cepheid GeneXpert system. In the majority of these cases, RT-qPCR cycle threshold (C_T) values were >38 (Table 1). However, at the end of October 2020, a mutation in the SARS-CoV-2 N gene was suspected when Xpert failed to amplify the N gene target in a specimen, despite giving a strong positive result (C_T =19.8) for the E gene. Subsequently, 3 more samples showed similar results in the next 2 months (Table 1). All of these samples were confirmed to be positive by a second test method (QIAstat-Dx respiratory SARS-CoV-2 panel; Qiagen). The study involves the secondary use of anonymous, residual pathological specimens that fall under the category “exempted” by the Sidra Medicine Institutional Review Board.

TABLE 1.

Summary of RT-qPCR and N gene sequencing data obtained from suspected N gene variants compared to the wild-type controls

Sample group	Test date in 2020	Sample ID	Cepheid Xpert test		Genotype or result of N gene sequencinga
			E gene CT	N gene CT
Control samples	03 November	Wt1	23.5	26.0	WT
	09 November	Wt2	19.5	21.8	WT
N gene-negative, low-titer samples	15 October	S1	38.6	Undetermined	ND
	17 November	S2	38.2	Undetermined	ND
	05 December	S3	39.0	Undetermined	ND
Suspected N gene mutants	27 October	Mt1	19.8	Undetermined	C29200A
	28 October	Mt2	34.3	Undetermined	Failed
	15 November	Mt3	26.8	Undetermined	C29200A
	05 December	Mt4	29.5	Undetermined	C29200A

^aWT, wild type; ND, not done.

We sequenced the entire N gene in these samples, along with two other samples that were positive for the N gene and E gene, by Sanger sequencing. cDNA was prepared using the Superscript IV RT cDNA synthesis kit (ThermoFisher). The N gene was amplified with 3 sets of primers (see Table S1 in the supplemental material) using Platinum Taq high-fidelity DNA polymerase (ThermoFisher). Sequencing was performed with a BigDye Terminator v3.1 cycle sequencing kit (ThermoFisher). All procedures were performed according to manufacturer’s instructions. Sequence data were analyzed in Geneious prime 2019.2.3. Sequencing was unsuccessful for 1 of the 4 suspected N gene variants. A point mutation (C29200A) was consistently detected in all three suspected variants but not in the SARS-CoV-2 consensus sequence (Fig. 1). A review of different SARS-CoV-2 target sequences that were recommended by the WHO revealed that the point mutation falls into the probe sequence of one of the CDC assays (2019_nCoV_N2) (https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf).

FIG 1.

DNA chromatogram alignment of the wild type and C29200A variants. Arrows at the top show the primers and the probe for the CDC 2019_nCoV_N2 assay. Dotted line shows the position of SNP C29200A.

Although the N gene target of the Cepheid Xpert assay is proprietary, the mutation in one of the CDC-developed assay targets suggest that a C29200A substitution may be responsible for the failure of amplification of the N gene by Xpert. Notably, none of these results were reported as false negative by the test platform because the E gene was amplified, and a discrepancy in the results for two targets warranted further investigation. Interestingly, a point mutation in the same genome position was independently reported from Germany in another study, but with a C29200T substitution. According to the study, 0.2% of the isolates in the EpiCoV database contain the C29200T mutation (4). However, using a BLAST search against the entire nucleotide collection in the NCBI, we were unable to find any reports of the C29200A mutation. Although only a small fraction of our SARS-CoV-2 isolates harbored this mutation, detection of the point mutation at 4 time points independent of each other suggests that there may be more cases in the community that remain to be detected. Apart from the Xpert assay, the C29200A mutation may also affect the performance of other commercial or laboratory-developed RT-qPCR tests for SARS-CoV-2 that are based on the same set of primers and probe used for the CDC’s 2019_nCoV_N2 assay. Our results provide further support for the possibility that novel SARS-CoV-2 variants may escape detection by nucleic acid amplification tests (NAAT), in particular if they are tested by assays based on single gene targets, and attest to the importance of having more than one PCR assay available for confirmation of ambiguous results.