CN113186340A - Primer group for rapidly detecting S gene point mutation of fragmented new coronavirus and application - Google Patents

Abstract

The invention discloses a group of primers for rapidly detecting S gene point mutation of fragmented new coronavirus and application thereof, wherein the primers comprise the following primer groups: a group of LDR primers LDR-1 and LDR-2 and a group of PCR primers LDR-UF and LDR-UR, wherein each primer is a single-stranded DNA molecule; the invention combines the ligase detection reaction and the polymerase chain reaction, can detect the point mutation contained in the highly degraded fragmented RNA with the length as low as 40nt, and the prior reverse transcription-fluorescence quantitative PCR technology can only detect the RNA template with the length of more than 100 nt.

Description

Primer group for rapidly detecting S gene point mutation of fragmented new coronavirus and application

Technical Field

The invention relates to the fields of biochemistry and molecular biology, in particular to a specific primer and a detection method for rapidly detecting S gene point mutation of fragmented new coronavirus.

Background

The disease (COVID-19) caused by the novel coronavirus (SARS-CoV-2) is a major public health emergency with the fastest transmission speed, the widest infection range and the greatest prevention and control difficulty since the establishment of new China, and is also a global pandemic infectious disease identified by the world health organization.

The new coronavirus belongs to single-stranded positive-sense RNA virus, is easy to mutate in the replication process, and researchers have issued nearly 8 ten thousand new coronavirus gene sequences at present. Although most genetic mutations do not affect viral virulence and infectivity, it has been reported that the D614G mutation in the S protein of the novel coronavirus significantly enhances the infectivity of the virus and reduces the susceptibility of the virus to the patient' S convalescent serum. How to rapidly distinguish these SNP sites in the detection of viral nucleic acid is always an important issue of interest to researchers.

The genetic material of the new coronavirus is single-stranded RNA, which is more unstable than double-stranded DNA. When the sampling, nucleic acid extraction and preservation processes are not operated properly, or nuclease, acid, alkali and other substances exist, the nucleic acid is easy to degrade and break into small fragments. The current main nucleic acid SNP detection means mainly comprise fluorescence quantitative PCR, a sequencing method and the like.

The mainstream detection means of nucleic acid SNP at present comprises a probe method fluorescent quantitative PCR and a sequencing method. The sequencing method is most accurate, but takes a long time, and is not favorable for clinical rapid detection. The probe method is used for detecting point mutation on RNA virus by fluorescence quantitative PCR, firstly virus RNA is reversely transcribed into cDNA, and then a pair of PCR primers designed according to the upstream and downstream of a mutation site and a Taqman-MGB fluorescent probe covering the mutation site are used for carrying out fluorescence quantitative PCR. Because the Taqman probe needs to be designed between two primers, the template length required by the probe method fluorescence quantitative PCR is generally not less than 100nt, and if the virus nucleic acid is damaged in the extraction and storage processes and the fragmentation degree is serious, the detection is difficult.

Disclosure of Invention

The invention aims to provide a specific primer and a detection method for rapidly detecting the S gene point mutation of fragmented new coronavirus by combining a Ligase Detection Reaction (LDR) technology with a fluorescent quantitative PCR technology aiming at the defects of the prior art, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: specific primers for rapidly detecting the fragmentized new coronavirus S gene point mutation comprise LDR primer groups LDR-1 and LDR-2 for detecting the new coronavirus S gene and PCR primer groups LDR-UF and LDR-UR, wherein each primer is a single-stranded DNA molecule; the LDR primer group comprises 2 primers, wherein the nucleotide base sequence of LDR-1 is SEQ ID No.1, and the nucleotide base sequence of LDR-2 is SEQ ID No. 2; the PCR primer set comprises 2 primers, wherein the nucleotide base sequence of LDR-UF is SEQ ID No.3, and the nucleotide base sequence of LDR-UR is SEQ ID No. 4.

The application of the primer group in the rapid detection of the fragmented new coronavirus S gene point mutation,

the specific detection method is as follows:

the method comprises the following steps: obtaining viral RNA;

step two: taking the RNA obtained in the first step as a template, adding the LDR primer pair LDR-1/LDR-2 of claim 1 to prepare LDR reaction mixed solution, and carrying out LDR reaction;

step three: performing a fluorescent quantitative PCR reaction on the reaction product of the second step by using the PCR primers LDR-UF and LDR-UR as described in claim 1, and then performing result judgment.

In the LDR reaction in the second step, the heat-resistant ligase used can be Taq ligase or Tth ligase.

The invention has the beneficial effects that: the invention can detect point mutation contained in highly degraded RNA with the length as low as 40-50nt, while the prior reverse transcription-probe method fluorescent quantitative PCR technology can only detect RNA template with the length more than 100 nt.

Drawings

FIG. 1 is a schematic diagram of the LDR-qPCR reaction of the present invention;

FIG. 2 is a design diagram of a LDR primer probe of the present invention;

FIG. 3 is a graph of LDR-qPCR amplification curves of different length RNA templates according to the present invention.

Note: A. template region 50nt is a perfect match to probe region (50 nt); B. the template region 50nt is moved upstream by 5nt relative to the probe region (50 nt); C. the template region 50nt is moved 10nt upstream relative to the probe region (50 nt); D. the template region 50nt and the opposing probe region (50nt) are moved downstream by 5nt to the right; E. the template region 50nt and the opposing probe region (50nt) are moved downstream by 10nt to the right; ntc (no template added).

Detailed Description

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention more readily understood by those skilled in the art, and thus will more clearly and distinctly define the scope of the invention.

The invention provides a technical scheme that: specific primers for rapidly detecting new coronavirus S gene point mutation comprise a group of LDR primers LDR-1 and LDR-2 and a group of PCR primers LDR-UF and LDR-UR, wherein each primer is a single-stranded DNA molecule; the nucleotide sequences are SEQ No.1, SEQ No.2, SEQ No.3 and SEQ No.4 in Table 1 respectively

The specific detection method is as follows:

the method comprises the following steps: obtaining virus RNA in a sample to be detected;

step two: taking the RNA obtained in the step one as a template, setting the S gene RNA of the wild-type new coronavirus as a contrast, respectively adding a primer pair LDR-1/LDR-2 to prepare LDR reaction mixed liquor, and carrying out LDR reaction;

step three: and (4) carrying out fluorescent quantitative PCR reaction on the reaction product of the second step by using a primer pair LDR-UF/LDR-UR, and then judging.

The LDR reaction mixed liquor in the second step comprises: heat-resistant DNA ligase, an RNA template, an LDR primer, a reaction buffer solution and deionized water; in the LDR reaction in the second step, the heat-resistant ligase can be Taq ligase or Tth ligase; in the LDR reaction solution in the second step, the working concentration of the primer can be 0.5-10 nmol/L; the LDR reaction procedure in the second step is as follows: firstly, 3 minutes at 95 ℃, 30 seconds at 95 ℃ and 4 minutes at 58 ℃; and secondly, repeating 30-40 cycles.

The evaluation mode in the third step is as follows: if the Ct value of the RNA of the sample to be detected is obviously lower than that of the wild type after LDR-qPCR reaction, the RNA of the sample is shown to have D614G mutation; otherwise, the RNA sample does not contain the D614G mutation.

Example 1:

new coronavirus S gene templates with the length of only 50nt were detected.

1. The method comprises the following operation steps:

(1) the recombinant plasmid containing the D614G mutation target segment of the new coronavirus S gene is transcribed in vitro to obtain an RNA segment with the length of 50nt and containing the D614G point mutation, and the RNA segment is used for simulating fragmented virus RNA.

(2) Preparing LDR reaction mixed liquor by the RNA template obtained in the step (1) and a primer pair LDR-1/LDR 2; LDR reactions were carried out using 50nt of the wild-type S gene RNA obtained in the same manner as in (1) as a control. The LDR reaction mixed liquid comprises: heat-resistant DNA ligase, an RNA template, an LDR primer pair, a 10X reaction buffer solution and deionized water. The amounts of the components added to the LDR reaction mixture are shown in Table 2. Then starting LDR reaction, wherein the reaction procedure is as follows: at 95 deg.c for 3 min, at 95 deg.c for 30 sec and at 58 deg.c for 4 min. Wherein, the step II is repeated for 35 cycles, and the specific reaction procedure is shown in Table 3.

TABLE 2 LDR reaction System

Components	Volume of
		High temperature resistant ligase	1μl
10 Xreaction buffer	2μl
		RNA template	1μl
LDR-1(1μmol/l)	1μl
		LDR-2(1μmol/l)	1μl
ddH2O	addto20μl

TABLE 3 LDR reaction procedure

(3) And (3) carrying out fluorescent quantitative PCR reaction detection on the reaction product of the step (2) by using primers LDR-UF and LDR-UR.

2. And (4) analyzing results:

the detection result is shown in figure 3A, the template containing the D614G mutation can be obviously amplified after being subjected to LDR-qPCR reaction, and the Ct value of the template is about 8 cycles lower than that of a wild type, which shows that the specific primer group can detect the D614G mutation in the highly degraded (fragment length of 50nt) new coronavirus S gene.

Example 2

The template length is smaller than the primer set, and the mutation can still be detected:

1. the method comprises the following operation steps:

(1) the recombinant plasmid containing the target fragment of the S gene of the new coronavirus was transcribed in vitro using different primers to obtain different RNA fragments with a length of 50nt containing the D614G point mutation, mimicking fragmented viral RNA that did not match perfectly with the probe region of the LDR primer (FIG. 2).

(2) And (2) preparing LDR reaction mixed liquor by using the RNA obtained in the step (1) as a template and a primer pair LDR-1/LDR2, and simultaneously performing LDR reaction by using 50nt wild S gene RNA as a control. The LDR reaction system and procedure are shown in tables 2 and 3.

(3) And (3) carrying out fluorescent quantitative PCR reaction detection on the reaction product of the step (2) by using primers LDR-UF and LDR-UR.

2. And (4) analyzing results:

the results of the experiments are shown in FIGS. 3B-E. Although the template and the primer probe region are not completely matched, the Ct value of an amplification curve taking the RNA containing the D614G mutation as the template is still 8 cycles lower than that of the wild type, which shows that when the specific primer group is used for detecting the D614 mutation in the fragmented new coronavirus S gene, the minimum detection template length can reach 40nt, which is far lower than the template length required by the fluorescence quantitative PCR of a common probe method.

The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims (3)

1. A group of primers for rapidly detecting the fragmentized new coronavirus S gene point mutation and application thereof are characterized by comprising LDR primer groups LDR-1 and LDR-2 for detecting the new coronavirus S gene and PCR primer groups LDR-UF and LDR-UR, wherein each primer is a single-stranded DNA molecule; the LDR primer group comprises 2 primers, wherein the nucleotide base sequence of LDR-1 is SEQ ID No.1, and the nucleotide base sequence of LDR-2 is SEQ ID No. 2; the PCR primer set comprises 2 primers, wherein the nucleotide base sequence of LDR-UF is SEQ ID No.3, and the nucleotide base sequence of LDR-UR is SEQ ID No. 4.

2. The use of the primer set of claim 1 for rapidly detecting point mutation of S gene of fragmented new coronavirus, comprising the steps of:

the specific detection method is as follows:

the method comprises the following steps: obtaining viral RNA;

step two: taking the RNA obtained in the first step as a template, adding the LDR primer pair LDR-1/LDR-2 of claim 1 to prepare LDR reaction mixed solution, and carrying out LDR reaction;

step three: performing a fluorescent quantitative PCR reaction on the reaction product of the second step by using the PCR primers LDR-UF and LDR-UR as described in claim 1, and then performing result judgment.

3. The use of the primer set according to claim 2 for rapidly detecting the point mutation of the S gene of the fragmented new coronavirus, wherein the primer set comprises the following components: in the LDR reaction in the second step, the heat-resistant ligase used can be Taq ligase or Tth ligase.

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