What is 2019-nCoV

The new coronavirus (2019-nCoV), also known as SARS-CoV-2, belongs to the genus β-coronavirus. The virus is a protein-enclosed single-stranded positive-strand RNA virus with an envelope. The particles are round or oval in diameter. Between 60-140nm, because the surface is uneven, its morphology looks like a crown under the electron microscope, so it is called coronavirus (Figure 1). The virus is the seventh coronavirus that can cause disease in humans (of the six viruses previously found, four are more common in the population and have lower pathogenicity, generally causing only mild respiratory symptoms similar to the common cold; the other two It is known as SARS coronavirus and MERS coronavirus), so it is called a new coronavirus, parasitic and infected vertebrates (including humans).

2019-nCoV

Why We Choose Nucleic Acid Detection

For pathogen detection, although studies have shown that CRISPR [1] and immune methods can also be used for virus detection, nucleic acid detection is currently a virus infection detection due to the mature, simple and rapid nucleic acid detection technology and high sensitivity and specificity. The most important method and “gold standard”. Fluorescence RT-PCR and gene sequencing are currently the only approved methods. Compared with the two, the former has a greater advantage, usually can detect dozens or even hundreds of nucleic acid samples in 1 hour. Therefore, many hospitals and medical examination offices currently use RT-qPCR.

The genetic material of the new coronavirus is RNA. If the RNA sequence of the new coronavirus is suspected to be detected in the patient’s sample, the patient may be infected with the new coronavirus. The most common method for detecting new coronavirus nucleic acids is fluorescent quantitative PCR. Since the new coronavirus is an RNA virus, the nucleic acid detection kit basically uses reverse transcription plus real-time fluorescent PCR to amplify the nucleic acid (RNA) of the new coronavirus, and at the same time detects the amplified products in real time through specific fluorescent probes. The products of different manufacturers will determine the positive judgment value of this product according to the performance of their own products.

The Principle of Nucleic Acid Detection Kits

Currently approved products are based on the open reading frame 1a/b (ORF1ab), envelope protein (Eve) and nucleocapsid protein (N) in the novel coronavirus genome (Figure 2) For selection, there are also a few commercial kits that simultaneously detect the spike glycoprotein (S) gene [2]. The detection principle of different products is basically the same, but the design of primers and probes are different. There are single target (ORF1ab), double target (ORF1ab, N protein), three targets (ORF1ab, N protein gene and E protein gene) detection and Interpret the difference. The new coronavirus transcript contains a large amount of sub genomic RNA [3]. The ORF1ab gene is transcribed from the full-length viral genome, so it only exists in the complete viral genome and can only be transcribed from the full-length viral genome, while the N gene is present in all Long genomic and other sub genomic RNA [4]. Therefore, during the transcription process of New Coronavirus, its N gene transcription synthesis amount is higher than that of ORF1ab. Especially for patients with asymptomatic infection or patients with low viral load in the recovery period, theoretically the content of ORF1ab gene transcribed and synthesized in patients is lower than that of N gene, so single-target nucleic acid detection reagents for ORF1ab gene only have low viral load Samples and virus mutants are prone to miss detection, and the detection of low-concentration viruses in the “Report on the Quality Evaluation of New Coronavirus Nucleic Acids in the National Laboratory” also proves this inference. The report shows that the sensitivity of different reagents to different target regions also differs: the quality evaluation samples are virus-like particles with ORF and N regions connected together, so the number of ORF and N regions is completely the same, but different reagents the positive rate of detection in each area is obviously different. Judging from the reagents with the number of results> 10, the positive rate in the N region is higher than that in the ORF region, and the lower the sample concentration, the more significant this phenomenon is [5]. In summary, the dual detection target products that simultaneously detect the N gene and the ORF1ab gene can better avoid the occurrence of missed detection caused by the above factors.

According to the New Coronavirus Pneumonia Laboratory Testing Technical Guidelines (Fifth Edition) issued by the National Health and Health Commission, to confirm a case as positive in the laboratory, it is necessary to meet the two target specificities of the new coronavirus ORF1ab and N gene in the same specimen Real-time fluorescence RT-PCR test results are positive, or single target positive patients re-sampling / another type of sample test is still single target positive [6][7], so the laboratory nucleic acid test for new coronavirus should include at least the most conservative And specific ORF1ab region and N gene.

Figure 2 SARS-CoV-2 genome structure diagram and common amplification locations (pictures from the network)

New coronavirus nucleic acid detection (Figure 3) mainly undergoes the following steps: specimen collection, specimen transportation, specimen reception, reagent preparation, specimen pretreatment, nucleic acid extraction and sample addition, nucleic acid amplification [7]. First of all, according to the kit instructions or “New Coronavirus Infected Pneumonia Laboratory Testing Technical Guide”, sample collection, sample types include pharyngeal swabs, nasal swabs, sputum, bronchial lavage fluid, alveolar lavage fluid, etc. Because RNA is easily degraded, use RNase-free swabs and RNase-free storage tubes when collecting samples. After receiving the sample, it should be tested immediately. If it cannot be tested in time, it should be packaged at 4 ℃ and completed within 24 hours; the specimen that cannot be tested within 24 hours should be stored at -70 ℃ or below. For nucleic acid extraction of samples, the nucleic acid extraction reagent should use the nucleic acid extraction kit recommended in the instructions of the amplification kit. Finally, fluorescence PCR nucleic acid detection, that is, fluorescence PCR instrument for amplification detection and analysis, according to the instructions to judge the negative result.

What Are We

Heal Force integrates R&D, manufacturing, sales and service, focusing on providing complete and reliable digital medical and laboratory solutions, with products in more than 100 countries and regions around the world. Thirty years into the medical field, Heal Force has been a large-scale medical device manufacturer and supplier in China.

Heal Force can provide different equipment configuration schemes according to different life science laboratories. Products include biological safety cabinets, clean benches, high/low-speed centrifuges, pure water equipment, incubators, PCR instruments, and low-temperature cold chain equipment. Heal Force has an excellent R&D team, a high-quality sales team and a comprehensive and experienced after-sales service team, which can provide users with first-class products and satisfactory user experience.

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