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Specific primer for novel coronary virus detection and rapid detection method

A coronavirus, detection method technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. and other problems, to achieve the effect of good sequencing quality, rapid and accurate detection

Active Publication Date: 2020-06-30
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT) +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The most rapid and effective nucleic acid detection method in the prior art is RT-PCR, and the specific technology used is the new coronavirus RT-PCR kit, but this method has certain defects. According to the RT-PCR system provided by the kit instructions, Add one to three pairs of primers with different specificities to check the specificity, and the detection area is ORFlab, N area and E area
Since the entire genome region of 2019-nCoV is not completely covered, its accuracy is affected to a certain extent, and RNA viruses are prone to mutation and recombination. There have been many cases of positive patients with negative RT-PCR tests
For differential diagnosis or a large number of samples to be tested, a large amount of reagents, consumables and time will be consumed when there are many test items. In addition, the resulting missed screening will also bring corresponding difficulties to subsequent diagnosis and prevention and control.

Method used

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  • Specific primer for novel coronary virus detection and rapid detection method
  • Specific primer for novel coronary virus detection and rapid detection method
  • Specific primer for novel coronary virus detection and rapid detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Such as figure 1 as shown, figure 1 It is a schematic flow diagram of the rapid detection method of the novel coronavirus; the rapid detection method of the novel coronavirus of the present invention comprises steps:

[0046] S1, inactivate the collected samples and extract viral RNA;

[0047] Virus inactivation treatment method: According to literature and guideline reports, coronavirus can be inactivated at 56°C for 30 minutes. Our preliminary tests show that the inactivation method of dry bath (oven or incubator) at 56°C for 30 minutes or 50°C for 60 minutes does not affect respiratory samples. Detection of RNA viruses. Preferably, respiratory samples (except for the purpose of cultivating microorganisms) are first dry bathed at 56°C for 60 minutes, allowed to stand for 10 minutes to room temperature (to avoid aerosols), and then processed.

[0048]Viral RNA extraction and quality inspection: The collected biological samples, including throat swabs, alveolar washi...

Embodiment 2

[0092] In this embodiment, for a highly suspected patient, but the three PCR results are all negative, such as Figure 4 as shown, Figure 4 It is the result graph of the three PCRs of the patient. However, when the rapid detection method of the present invention is used for detection, a large number of novel coronavirus genome sequences are observed in the patient's extracted samples, such as Figure 5 as shown, Figure 5 A map of the genome sequence alignment of the sample extracted for the patient. Figure 5 The first row is the reference genome of the new coronavirus, and a large number of high-coverage and uniform alignment sequences appear at the bottom. This result is positive through serological IgM test samples, which can verify that the patient is carrying the new coronavirus. As shown in Table 2, Table 2 is a list of the virus detected in this patient.

[0093] Table 2 List of detected viruses

[0094]

[0095] Such as Image 6 , Figure 7 as shown, Imag...

Embodiment 3

[0107] The present invention can be applied to the sample DNA comparison results at the same time, as shown in Table 5, Table 5 is the detection list of the Neisseriaelongata, Actinomycesgraevenitzii and Prevotellamelaninogenica sequences found in the sample in Example 2, which may be related to the actual operation of the sample Sometimes there is a small amount of pollution.

[0108] Table five

[0109]

[0110] The detected results of the present invention can be compared with homologous sequences of other species. Such as Figure 14 as shown, Figure 14 It is the result of simultaneous comparison between the sample of Example 2 and the new crown and SARS. The READs compared to the new crown are more than the READs compared to the SARS. At the same time, most of the READs compared to the SARS are also compared to the new crown. This shows that SARS There are many similarities with the pathogenic region of the new crown.

[0111] Among all the samples in Example 2, th...

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Abstract

The invention discloses a specific primer for novel coronary virus detection and a rapid detection method. The method comprises the following steps of inactivating a collected sample, and extracting virus RNA; carrying out inversion on virus RNA which is extracted in the step S1 and qualified through quality testing to perform cDNA synthesis; adopting the specific primer for fragment amplificationto form a PCR reaction product sample; identifying the PCR reaction product sample, and constructing a sequencing library; and carrying out a nanopore sequencing operation on the sequencing library.The method is high in sequencing quality, and when the sample obtained by the method is applied to novel coronary virus genome comparison, reads comparison rate as high as 94.16% can be realized, so that determination of the infection condition of a patient can be assisted, and rapid accurate detection of novel coronary virus can be realized.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a specific primer and a rapid detection method for detection of novel coronavirus. Background technique [0002] The novel coronavirus (2019-nCoV) infection is currently highly transmissible and has a high fatality rate, which is a major public health issue of national concern. At the same time, it is also the epidemic season of other respiratory viruses, which has a huge impact on clinical diagnosis and differential diagnosis. [0003] In view of the high prevalence, distribution and widespread nature of coronaviruses, the diversity of genes and frequent recombination of genomes, and the increasing interaction between humans and animals, it is very likely that new coronaviruses will appear periodically in humans, Therefore, there is an urgent need to study methods for its rapid detection. [0004] The most rapid and effective nucleic acid detection method in the prior art i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701Y02A50/30
Inventor 叶枫李征途余乐陈灵丹卢文菊刘婷婷李寅虎赵志强李少强占扬清成静陈莉延
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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