CN114645101A

CN114645101A - Multi-fluorescence detection primer probe set and kit for typing new coronavirus Ormcken variant strain

Info

Publication number: CN114645101A
Application number: CN202210415311.9A
Authority: CN
Inventors: 付建光; 艾静; 邓斐; 樊欢; 王慎骄; 余慧燕; 鲍倡俊; 朱立国
Original assignee: Jiangsu Center For Disease Control And Prevention (jiangsu Institute Of Public Health)
Current assignee: Jiangsu Center For Disease Control And Prevention (jiangsu Institute Of Public Health)
Priority date: 2022-04-20
Filing date: 2022-04-20
Publication date: 2022-06-21
Anticipated expiration: 2042-04-20
Also published as: CN114645101B

Abstract

The invention provides a multiple fluorescence detection primer probe group and a kit for typing new coronavirus Ormcken variant strains. The multi-fluorescence detection primer probe group and the kit for typing new coronavirus Ormcken variant strains have the following technical effects: 1. the cost is low, and the total reagent cost of a single specimen is 12 yuan. 2. The operation is simple and convenient, the result is clear, the universal reagent and the primer probe working solution can be used for on-machine detection, and the infection of the Ormcken variant strain BA.1 type, BA.2 type and other Ormcken variant strains can be judged simultaneously, and the total detection time is about 1.5 hours. Simple operation process, conventional fluorescence detector equipment and basic unit can be developed. 3. The sensitivity is high, and the method can be applied to environmental sample detection. High-throughput sequencing of environmental samples is difficult to perform due to low viral load, and the method of the invention can also be characterized to an Onckronk specific type when the environmental samples are detected.

Description

Multi-fluorescence detection primer probe set and kit for typing new coronavirus Ormcken variant strain

Technical Field

The invention relates to a multiple fluorescence detection primer probe group and a kit for typing new coronavirus Ormcken variant strains, and belongs to the technical field of biology.

Background

Research shows that variation speed of variation strain of Ormckron is faster than that of wild strain or other VOC, four main types including BA.1, BA.2, BA.3 and BA.1.1 exist at present, and the main types causing spread and prevalence in China are BA.1 and BA.2. The prevention and control tracing at the first time when the new crown epidemic situation appears becomes a normal state, but the virus type is unknown, and the joint seal and the source of the virus are difficult to effectively judge, so that the prevention and control tracing is passive. However, the current mainstream Ormcken typing detection method is high-throughput sequencing, and has high economic cost and long time consumption. Meanwhile, many basic detection units do not have high-throughput sequencers, so that the detection of the Ormcken typing is difficult to carry out in the first time, and the precious prevention and control time and the establishment of prevention and control strategies are delayed.

The existing Onckrojon typing fluorescence detection kit is used for detecting specific several mutation sites, and only one genotype can be detected, so that the cost is high and the operation is complex.

The applicant filed an invention patent application with patent application number "2022101630826" on 22/2/2022 with the title "a multiple fluorescence detection primer probe set and kit for the Ormckh variant of new coronavirus". The primer probe set and the kit are mainly used for simultaneously judging whether to be infected by SARS-CoV-2 and whether to be infected by the Ormcken variant strain, and do not relate to the typing of the Ormcken variant strain of the new coronavirus.

Chinese patent with patent application No. 202111618758.8 entitled "composition, kit, method for detecting SARS-CoV-2 variant and use thereof" provides a kit comprising the composition, use of the composition, and a method for detecting and typing SARS-CoV-2 variant. In this patent, the expression typing is used to distinguish other types of new coronavirus from the Onckrojon type, i.e., the patent can distinguish whether the variant is the Oncojon type, but cannot determine which type of virus is the Oncojon type.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a multiple fluorescence detection primer probe set and a kit for typing new coronavirus Ormcken variant strains.

The present inventors designed a multiplex fluorescence detection primer probe set for typing an Oncuronn variant, which can simultaneously detect two gene fragments (S gene and N gene) of SARS-CoV-2, the S gene for determining BA1 infection and BA2 infection, and the N gene for determining the infection of the Oncuronn variant. All primer probes for detection were designed by the inventors themselves. The Omicron-F primer is positioned at the 28316-28335 nucleotide site of the whole genome, the Omicron-R primer is positioned at the 28435-28454 nucleotide site of the whole genome, and the Omicron-Probe primer is positioned at the 28362-28381 nucleotide site of the whole genome; the BA1-F primer is located at the 22191-22211 nucleotide site of the whole genome, the BA1-R primer is located at the 22327-22348 nucleotide site of the whole genome, and the BA1-Probe primer is located at the 22213-22234 nucleotide site of the whole genome; the BA2-F primer is located at the 21583-21607 nucleotide site of the whole genome, the BA2-R primer is located at the 21710-21733 nucleotide site of the whole genome, and the BA2-Probe primer is located at the 21620-21647 nucleotide site of the whole genome.

In the invention, the nucleotide sequence of the multiplex fluorescence detection primer probe group used for typing Ormcken variant strains of new coronavirus is shown in the following table:

wherein, the first and the second end of the pipe are connected with each other,

the fluorescent probe is an oligonucleotide probe with a fluorophore attached to the 5 'end of the probe and a quencher attached to the 3' end of the probe. The fluorescent group for detecting BA.1 is FAM, and the quenching group is DBQ 1. The fluorescent group for detecting BA.2 is VIC, and the quenching group is DBQ 1. The fluorophore for detection of Ormckhen infection was CY5 and the quencher was BHQ 2.

The invention also provides a preparation method of the primer probe set working solution, which comprises the following steps:

1) the synthesized primer probe dry powder and the RNase-free water are prepared into a working concentration of 10 micromole/liter.

2) Primer probe sets to be used for detection of infection by oleckjon BA1, BA2 and oleckjon were as follows upstream primer: a downstream primer: the volume ratio of the probes is 2:2:1, and three probe primer mixed solutions are prepared respectively; in the present invention, the upstream primer: a downstream primer: the volume ratio of the probe added was 400:400:200 (. mu.L).

3) And finally, mixing the three probe primer mixed solutions in equal volume to prepare a primer probe working solution.

During detection, the universal multiple fluorescence detection reagent and the primer probe working solution can be used for on-machine detection to judge the infection type of the Ormcken variant strain.

The invention also provides a multiple fluorescence detection kit for typing the Ormckh variant strain of the new coronavirus, which comprises the multiple fluorescence detection primer probe group for typing the Ormckh variant strain of the new coronavirus.

Further, the Kit also comprises a multiplex fluorescence detection reagent, and the multiplex fluorescence detection reagent can be a market universal multiplex fluorescence detection reagent, such as a HiScript II U + One Step qRT-PCR Probe Kit reagent produced by Nanjing Novozam.

The present invention was made in further detail based on the research of the invention patent application No. 2022101630826 entitled "multiple fluorescence detection primer probe set and kit for Ormckro variant strain of New crown virus" filed on 22/2/2022.

The invention concept of the invention is as follows:

and designing a PCR primer and a fluorescent Taqman probe aiming at the gene conserved sequence of the specific Ormcken variant strain. A nucleotide probe with two ends marked with fluorescent dye groups is added on the basis of conventional PCR, wherein the fluorescent group is arranged at the 5 'end, the quenching group is arranged at the 3' end, and the fluorescent group and the quenching group form an energy transfer structure. When the probe is complete, the fluorescent group is inhibited by the quenching group and does not generate fluorescence, when the probe is combined with the target sequence, the upstream primer extends to the position, the probe is hydrolyzed under the action of exoenzyme, and the fluorescent signal of the fluorescent group is released and collected and detected by an instrument, so that the existence of the target sequence is prompted. The inventors added a fluorescent probe primer sequence for the N gene to determine the infection of Onckrojon and a fluorescent probe primer sequence for the S gene to determine the infection of the Onckrojon types BA.1 and BA.2, thereby forming a combination, and using the real-time property, high sensitivity and good specificity of the fluorescent PCR technology, the infection type of the variation strain of Onckrojon can be determined intuitively and rapidly at the end of the reaction and even before the end. In addition, FAM and VIC are selected as fluorescent groups of the S gene, a double-quenching probe DBQ1 is selected as a quenching group instead of BHQ1, namely, the quenching group is additionally added in the middle of a conventional probe, and the fluorescence intensity after amplification is greatly improved on the premise of ensuring low background signals.

In contrast to the prior art CN 202111618758.8, the typing method of the present invention firstly distinguishes whether the variant strain is an Onckhorden type, and then further determines which subtype, such as BA.1 or BA.2, is the Onckhorden type. The current epidemic strain is mainly BA.2, a small amount of BA.1 sporadic infection exists, the patent with the patent application number of '202111618758.8' cannot distinguish the two strains, and the patent can realize the purpose, so the two strains have essential difference.

The multi-fluorescence detection primer probe group and the kit for typing new coronavirus Ormcken variant strains have the following technical effects:

1. the manufacturing cost is low.

In the primer synthesis, only the price of the probe is slightly high, the three genes consist of 6 primers and 3 probes, each primer only needs dozens of yuan, the number of the probes is about 1500 yuan, the total synthesis cost is about 5000 yuan, 1200 specimens can be detected by calculating the lowest synthesized 1 OD, and the cost of each specimen is about 4 yuan. The kit is a universal multiple fluorescence detection kit, the 100 persons are about 800 yuan, and the cost of each specimen is about 8 yuan. Therefore, the overall reagent cost for a single specimen is 12 dollars. The existing marketed Oncurong fluorescence typing detection kit is used for detecting specific several variable sites, the cost is high, 50 people share about 10000 yuan, and the cost of a single specimen is 200 yuan, which is 16 times of that of the invention. In addition, the high-throughput sequencing method is higher in cost, and the reagent cost of a single specimen is 2000 yuan. In the present invention, HiScript II U + One Step qRT-PCR Probe Kit produced by Nanjing Novozam was selected. The kit is 100 parts, and the result is not obviously changed when the inventor reduces one third of the original amount, so that the kit for 100 parts can detect 300 samples, and the actual cost is lower.

2. High sensitivity, good specificity and good repeatability, and can be applied to environmental sample detection

The present invention is designed based on the sequences of all Onckrojon genotypes that are present globally, and theoretically, infection with all types of Onckrojon variant strains can be detected. In practical application, the inventors tested all the ormikrong variants of Jiangsu (including three types BA.1, BA.2 and BA.1.1), and used Alpha (B.1.1.7) variants, Delta (B.1.617.2) variants and other viruses as controls, and showed 100% sensitivity and specificity. When the weekly new coronavirus specimen is subjected to rechecking, the result is found to be completely consistent with the sequencing result, and the good stability and repeatability are shown. In addition, the environment sample is detected in the place which can best show the advantage of sensitivity. The environmental sample amount is large, the virus carrying capacity is low, and great difficulty is caused to the detection work. Environmental sample is difficult to carry out high-throughput sequencing due to low virus load, but the current situation is that the infected places basically have environmental pollution, and whether the infection of the Onckronen variant strain is provided or not cannot be provided due to the failure of sequencing. The method can detect the infection of the Ormcken variant strain and the specific type when detecting the environmental sample, the detection result is completely consistent with the epidemiology traceability investigation result, and the sensitivity and the specificity are both 100 percent.

Drawings

FIG. 1 shows the results of the specimen examination of Ormcken variant BA.1 type patient.

FIG. 2 shows the results of the specimen examination of Ormcken variant BA.2 type patients.

FIG. 3 shows the results of the sample test of Ormcken variant strain BA.1.1 type patient.

FIG. 4 shows the results of environmental sample tests (infection with Ormcken variant strain BA.1).

FIG. 5 shows the results of specificity verification (Alpha variant B.1.1.7, Delta variant B.1.617.2, influenza virus, enterovirus, rotavirus and norovirus).

FIG. 6 shows the comparison of the single quenching probe BHQ1 and the double quenching probe DBQ 1.

FIG. 7 shows the results of a comprehensive comparison of samples of patients with the type-BA.1 and type-BA.2 Onckrojon variants.

Detailed Description

The following are specific embodiments of the present invention and are further described with reference to the drawings, but the present invention is not limited to these embodiments.

Example 1

1. Designing a primer probe:

the primers for typing and detecting the mutant Onckrojon are designed by the inventor by downloading the sequences of the Onckrojon of all genotypes (including four genotypes of BA.1, BA.2, BA.3 and BA.1.1), comparing and analyzing 26 gene fragments, and finally designing 110 pairs of primer probes in the S region and 10 pairs of primer probes in the N region. The primer probes were subjected to specific alignment analysis by NCBI BLAST online database. The screening of primers with high PCR efficiency, high sensitivity, good specificity and good stability finally obtains 2 pairs of primers and corresponding probe sequences of the S region which can be used for judging the infection of the type of Onckronen BA.1 and BA.2, and 1 pair of primers and corresponding probe sequences of the N region which can be used for judging the infection of the Onconen. Meanwhile, the quenching group of the DNA is replaced by DBQ1 from BHQ1, so that the stability and the amplification efficiency of the DNA are improved, and the background noise is reduced.

TABLE 1 Multi-fluorescence detection primer Probe sequences for Ormcken variant typing

Name (R)	Sequence of	Decoration
			BA1-F	TTA TAG TGC GTG AGC CAG AAG
BA1-R	AGC TGT CCA ACC TGA AGA AGA A
			BA1-Probe	TCT CCC TCA GGG TTT TTC GGC T	5’FAM/DBQ1 3’
BA2-F	ATT GCC ACT AGT CTC TAG TCA GTG T
			BA2-R	AGG TAA GAA CAA GTC CTG AGT TGA
BA2-Probe	ACC AGA ACT CAA TCA TAC ACT AAT TCT T	5’VIC/DBQ1 3’
			Omicron-F	TGC ACT CCG CAT TAC GTT TG
Omicron-R	AGT GAG AGC GGT GAA CCA AG
			Omicron-Probe	AAC CAG AAT GGT GGG GCG CG	5’CY5/BHQ2 3’

2. Kit for multi-fluorescence detection of Ormcken variant typing

The Kit for multiplex fluorescence detection of the Onckrojon variant strain comprises the primer Probe set and multiplex fluorescence detection reagents, wherein the multiplex fluorescence detection reagents can be selected from the multiplex fluorescence detection reagents currently used in the market, and the Kit for HiScript II U + One Step qRT-PCR Probe produced by Nanjing Novozam is selected by the inventor.

The primer probe group is used as primer probe working solution by the following preparation method:

1) the primer probe synthetic dry powder is added with RNase-free water to prepare a working concentration of 10 micromoles/liter.

2) According to an upstream primer: a downstream primer: the probe and primer mixture was prepared at a volume ratio of 2:2:1, and the volume ratio in the present invention was 400:400:200(μ L).

3) And finally, mixing the three mixed solutions in equal volume to prepare the primer probe working solution.

During detection, the universal multiple fluorescence detection reagent and the prepared primer probe working solution can be used for on-machine detection, and whether the Ormcken variant strain is infected or not and the infection type can be judged at the same time.

3. The detection process is as follows:

3.1 preparation of the system

The experimental system formulation was carried out in a system formulation laboratory. The multiplex fluorescence detection Kit currently used in the market can be selected, and the HiScript II U + One Step qRT-PCR Probe Kit produced by Nanjing Novozam is selected by the inventor. The system formulation is shown in table 2:

TABLE 2PCR reaction System

Component name	Adding volume (mu L)/per part of human
		2×One Step U⁺Mix	5
One Step U⁺Enzyme Mix	0.5
		Dye	0.2
Primer probe working solution	2.4
		H₂O	1.9
Total volume	10

After the system is prepared, the mixture is shaken, evenly mixed and centrifuged, and the mixture is subpackaged into PCR reaction tubes according to 10 mu L/person.

3.2 sample handling

The sample nucleic acid to be tested, the positive control and the negative control are added into the prepared PCR reaction tube in a sample adding chamber, the volume is respectively 2.5 mu L, the final volume is 12.5 mu L, a tube cover is tightly covered, and the tube cover is vibrated and centrifuged. Wherein the sample is the nucleic acid of the Ormcken variant strain of the new coronavirus owned by the laboratory of the inventor, the types comprise three types of Ormcken variant strains BA.1, BA.2 and BA.1.1, and the source of the sample comprises a patient sample and an external environment sample. In addition, other new coronavirus types for specific detection include Alpha variant B.1.1.7 and Delta variant B.1.617.2, influenza virus, enterovirus, rotavirus, norovirus and other nucleic acid samples. The positive control is Ormcken variant strain BA.1 and BA.2 nucleic acid which are successfully sequenced. Negative control was rnase-free water.

3.3PCR amplification

And (3) putting the PCR reaction tube into an ABI QuantStudio Q5 fluorescent quantitative PCR instrument for amplification detection without ROX correction. Fluorophores are FAM, VIC and CY5, respectively. The cycle parameter settings are shown in table 3:

TABLE 3 reaction procedure

3.4. Analysis of results

The negative control showed no fluorescence curve, and the positive control showed three smooth curves. The specimen fluorescence curve is smooth, and the cycle number (Ct value) is less than 38, so that the specimen is judged to be positive, otherwise, the specimen is negative. When one of FAM and VIC has Ct less than or equal to 38 and CY5 Ct more than 38, the detection result is invalid. The specific determination results are shown in table 4:

TABLE 4 determination of results

(1) The result of the sample test of the Ormkjon variant strain BA.1 type patient shows that both FAM and CY5 channels show curves and Ct is less than or equal to 38, which indicates SARS-CoV-2 infection and is the Ormkjon variant strain. See FIG. 1

(2) The result of the sample test of the Ormcken variant strain BA.2 type patient shows that the VIC and CY5 channels have curves and Ct is less than or equal to 38, which indicates that the strain is SARS-CoV-2 infection and is the Ormcken variant strain. See FIG. 2

(3) The result of sample test of the Ormckhen variant strain BA.1.1 type patient shows that both FAM and CY5 channels show curves and Ct is less than or equal to 38, which indicates SARS-CoV-2 infection and is the Ormckhen variant strain. See FIG. 3

(4) The results of environmental sample tests (infection of Ormcken variant BA.1), curves appeared in both FAM and CY5 channels, and Ct is less than or equal to 38, indicating that the strain is SARS-CoV-2 infection and is an Ormcken variant. See FIG. 4

(5) Specificity verification (Alpha variant B.1.1.7 type, Delta variant B.1.617.2 type, influenza virus, enterovirus, rotavirus and norovirus), FAM, VIC and CY5 channels have no curves, which indicates that the primer combination does not react with other new coronavirus types and other virus nucleic acids. See FIG. 5

(6) Compared with a double-quenching probe DBQ1, the single-quenching probe BHQ1 and the double-quenching probe DBQ1 show that the curve appears in both the channel VIC and the channel CY5, and Ct is less than or equal to 38, which indicates that the strain is an Onckronk variant strain BA.2 type infection, but obviously, the curve of the double-quenching probe is smoother, the curve difference of the two channels is smaller, and the background noise is lower. See FIG. 6

(7) The results of the comprehensive comparison of the sample of the Ormck Ron variant strain BA.1 and the sample of the Ormck Ron variant strain BA.2 show that the sample of the Ormck Ron variant strain BA.1 presents the curve of FAM and CY5 channels and Ct is less than or equal to 38, which indicates SARS-CoV-2 Ormck Ron variant strain BA.1 infection; the sample VIC and CY5 channels of the Oromken variant strain BA.2 have curves, and the Ct is less than or equal to 38, which indicates that the sample is infected by the SARS-CoV-2 Oromken variant strain BA.2. See FIG. 7

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

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Claims

1. A multiplex fluorescence detection primer probe set for typing of an olmckinson variant of new coronavirus, wherein the nucleotide sequence of said primer probe set is as shown in the following table:

name (R) Sequence of SEQ ID NO BA1-F TTA TAG TGC GTG AGC CAG AAG 1 BA1-R AGC TGT CCA ACC TGA AGA AGA A 2 BA1-Probe TCT CCC TCA GGG TTT TTC GGC T 3 BA2-F ATT GCC ACT AGT CTC TAG TCA GTG T 4 BA2-R AGG TAA GAA CAA GTC CTG AGT TGA 5 BA2-Probe ACC AGA ACT CAA TCA TAC ACT AAT TCT T 6 Omicron-F TGC ACT CCG CAT TAC GTT TG 7 Omicron-R AGT GAG AGC GGT GAA CCA AG 8 Omicron-Probe AAC CAG AAT GGT GGG GCG CG 9

The fluorescent probe is an oligonucleotide probe, the 5 'end of the probe is connected with a fluorescent group, and the 3' end of the probe is connected with a quenching group.

2. The primer probe set according to claim 1,

the 5 'end of the probe for detecting BA1 is connected with a fluorescent group FAM, and the 3' end is connected with a quenching group DBQ 1;

the 5 'end of the probe for detecting BA2 is connected with a fluorescent group VIC, and the 3' end is connected with a quenching group DBQ 1;

the 5 'end of the probe for detecting Omicron is connected with a fluorescent group CY5, and the 3' end is connected with a quenching group BHQ 2.

3. The method for preparing a primer probe working solution comprising the primer probe set according to claim 1 or 2, comprising the steps of:

1) adding RNA-free enzyme water into the synthetic dry powder of each primer and probe to prepare the working concentration of 10 micromoles/liter;

2) three primer probe sets for detection of BA1, BA2 and Omicron, according to the upstream primer: a downstream primer: the volume ratio of the probes is 2:2:1, and three probe primer mixed solutions are prepared respectively;

4. A multiplex fluorescence detection kit for typing of oncokrron variants of new coronavirus comprising a primer probe set according to claim 1 or 2.

5. The kit of claim 4, further comprising a multiplex fluorescence detection reagent.

6. The Kit of claim 5, wherein the multiplex fluorescence detection reagent is HiScript II U + One Step qRT-PCR Probe Kit reagent of Nanjing Novozam.