What do 'n gene' (nucleocapsid gene) and 'ORF' (Open Reading Frame) refer to in COVID-19 testing?

From the Guidelines

In COVID-19 testing, the N gene (nucleocapsid gene) and ORF (Open Reading Frame) are crucial genetic targets used to detect SARS-CoV-2 virus, with the N gene encoding the nucleocapsid protein and ORF1ab being a commonly targeted region due to its large size and essential role in viral replication. The N gene is highly conserved across coronaviruses, making it a reliable target for COVID-19 detection, as noted in the study by 1. PCR tests often target this gene because it tends to be stable and less prone to mutations. Open Reading Frames are sections of genetic code that can be translated into proteins, with SARS-CoV-2 containing multiple ORFs.

The use of multiple genetic targets, including both the N gene and various ORFs, is a strategy employed by testing laboratories to increase test accuracy and reduce false negatives, especially as the virus mutates over time, as discussed in 1. This multi-target approach helps ensure that tests remain effective even when certain parts of the viral genome change through mutation. The recent guidelines from 1 emphasize the importance of using standard NAAT (nucleic acid amplification tests) for diagnosing COVID-19, which often target these genetic regions.

Key points about the N gene and ORF in COVID-19 testing include:

• The N gene encodes the nucleocapsid protein, which packages the viral RNA and is highly conserved across coronaviruses.
• ORF1ab is a commonly targeted region in COVID-19 tests due to its large size and essential role in viral replication.
• Using multiple genetic targets, including both the N gene and various ORFs, can increase test accuracy and reduce false negatives.
• The strategy of targeting these genetic regions is supported by recent guidelines from 1, which recommend the use of standard NAAT for diagnosing COVID-19.

From the Research

Definition of 'n gene' and 'ORF' in COVID-19 Testing

• The 'n gene' refers to the nucleocapsid gene, which is one of the genes used as a target for detection of SARS-CoV-2 in COVID-19 testing 2, 3.
• 'ORF' stands for Open Reading Frame, which is a portion of a DNA sequence that has the potential to encode a protein 2, 4.
• In the context of COVID-19 testing, ORF1a and ORF1ab are specific open reading frames that have been targeted for detection of SARS-CoV-2 genomic RNA 2, 5, 6.

Role of 'n gene' and 'ORF' in COVID-19 Testing

• The nucleocapsid (N) gene and spike (S) protein are commonly used targets for detection of SARS-CoV-2, but they may not be suitable for estimating the infectivity of a specimen due to the presence of subgenomic RNAs 2.
• The detection of ORF1a gene has been proposed as a more rational indicator for the possible infectivity of clinical specimens, as it specifically detects viral genomic RNA 2.
• The dynamic changes of ORF1ab and N gene Ct values have been analyzed in COVID-19 patients to understand the natural history of the virus infection and the time to resolution of Omicron infection 5.

Studies on 'n gene' and 'ORF' in COVID-19 Testing

• A study compared the performance of different RT-qPCR master mixes on the detection of SARS-CoV-2 using primer/probe assays targeting the N gene, and found that specific assays can perform well regardless of the mastermix used 3.
• Another study found that a sequence from the SARS-CoV-2 orf1b gene is present in the human NTNG1 gene, and suggested that this may be relevant to the treatment of COVID-19 4.
• A study analyzed the early evolutionary trends of SARS-CoV-2 and found that positive selection of ORF1ab, ORF3a, and ORF8 genes drives the early evolutionary trends of the virus 6.