CN115896356A

CN115896356A - Reagent for detecting nucleic acid of novel coronavirus, influenza A virus and influenza B virus and application of reagent

Info

Publication number: CN115896356A
Application number: CN202310002210.3A
Authority: CN
Inventors: 李小锋; 李晨阳; 刘小翠
Original assignee: Guangdong Hexin Health Technology Co ltd; Guangzhou National Laboratory
Current assignee: Guangdong Hexin Health Technology Co ltd; Guangzhou National Laboratory
Priority date: 2023-01-03
Filing date: 2023-01-03
Publication date: 2023-04-04

Abstract

The invention discloses a reagent for detecting nucleic acid of novel coronavirus, influenza A and influenza B virus and application thereof. The reagent for detecting the nucleic acid of the novel coronavirus, the influenza A virus and the influenza B virus has high sensitivity, strong specificity and short detection time, has no cross reaction with various other common respiratory pathogens, and has higher reference value for auxiliary diagnosis and clinical medication guidance of the common influenza virus and the novel coronavirus. Meanwhile, the invention also overcomes the defect that the nucleic acid detection kit needs refrigeration or frozen transportation and storage by adopting the fluorescence detection and freeze-drying technology, can be transported at normal temperature, saves the cost and has long storage time.

Description

Reagent for detecting nucleic acid of novel coronavirus, influenza A virus and influenza B virus and application of reagent

Technical Field

The invention relates to the technical field of virus detection, in particular to a reagent for detecting nucleic acid of novel coronavirus, influenza A virus and influenza B virus and application thereof.

Background

At present, the real-time fluorescence quantitative PCR technology is the most widely applied molecular detection technology, and the technology not only realizes the quantification of the nucleic acid template, but also has the characteristics of high sensitivity, strong specificity, high automation degree and the like.

Patent 202010205841.1 discloses a composition, a kit and a method for detecting and typing three viruses causing respiratory tract infection in one tube simultaneously by combining a component composition containing the composition with a fluorescent probe method, and the kit, the method and the use thereof.

Therefore, a novel method for detecting coronavirus, influenza a virus and influenza b virus, which has high sensitivity, strong specificity, wide coverage and simple operation, is needed.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. To this end, the invention proposes a reagent for detecting nucleic acids of novel coronaviruses, influenza A and/or influenza B viruses.

The invention also provides a kit with the reagent.

The invention also provides an application of the reagent or the kit.

According to one aspect of the present invention, there is provided a reagent for detecting a novel coronavirus, influenza a and/or influenza b virus nucleic acid, the reagent comprising a first primer combination for detecting influenza a virus, a second primer combination for detecting influenza b virus, a third primer combination for detecting a novel coronavirus;

wherein the first primer combination comprises a primer group with a sequence shown as SEQ ID NO.1 and SEQ ID NO.2, a primer group with a sequence shown as SEQ ID NO.13 and SEQ ID NO.14 or a primer group with a sequence shown as SEQ ID NO.16 and SEQ ID NO. 17;

the second primer combination comprises a primer group with a sequence shown as SEQ ID NO.4 and SEQ ID NO.5 or a primer group with a sequence shown as SEQ ID NO.19 and SEQ ID NO. 20;

the third primer combination comprises a primer group with a sequence shown as SEQ ID NO.7 and SEQ ID NO.8 or a primer group with a sequence shown as SEQ ID NO.22 and SEQ ID NO. 23.

In some embodiments of the invention, a primer group with a sequence shown in SEQ ID NO.7 and SEQ ID NO.8 is used for detecting the N gene of the novel coronavirus.

In some embodiments of the invention, a primer set having a sequence shown in SEQ ID NO.22 or SEQ ID NO.23 is used for detecting the ORF1ab gene of the novel coronavirus.

In some embodiments of the invention, the influenza a virus comprises influenza a seasonal H1N1, seasonal H3N2, H7N9, new influenza a H1N1 and H5N1.

In some embodiments of the invention, the influenza b virus comprises influenza b virus type Yamagata or Victoria.

In some embodiments of the invention, the reagents further comprise a probe sequence.

In some embodiments of the invention, the probe is selected from one or more of SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 15, SEQ ID NO 18, SEQ ID NO 21 and SEQ ID NO 24.

In some embodiments of the invention, the probe is selected from one or more of SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9.

In some embodiments of the invention, the probe sequence shown in SEQ ID NO. 3 is a fluorescent sequence matched with the primer group shown in SEQ ID NO.1 and SEQ ID NO. 2; the probe sequence shown in SEQ ID NO. 6 is a sequence matched with the nucleic acid molecules shown in SEQ ID NO.4 and SEQ ID NO. 5; the probe sequence shown in SEQ ID NO. 9 is a sequence matched with the nucleic acid molecules shown in SEQ ID NO.7 and SEQ ID NO. 8; the probe sequence shown in SEQ ID NO. 15 is a sequence matched with the nucleic acid molecules shown in SEQ ID NO.13 and SEQ ID NO. 14; the probe sequence shown in SEQ ID NO. 21 is a fluorescent sequence matched with the nucleic acid molecules shown in SEQ ID NO.19 and SEQ ID NO. 20; the probe sequence shown in SEQ ID NO. 24 is a fluorescent sequence matched with the nucleic acid molecules shown in SEQ ID NO.22 and SEQ ID NO. 23.

In some embodiments of the invention, the 5' end of the probe comprises a fluorescent reporter group, including any one of FAM, texas Red, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY5, and VIC.

In some embodiments of the present invention, the 5' end of the probe in the first primer set-third primer combination comprises different fluorescent reporter groups.

In some embodiments of the invention, the 3' end of the probe comprises a fluorescence quenching group, including any one of MGB, BHQ-1, BHQ-2, and BHQ-3.

In some embodiments of the invention, the reagents further comprise a detection reagent for an internal reference gene.

In some embodiments of the invention, the reference gene is β -Globin or GAPDH.

In some embodiments of the present invention, the detection reagent for the reference gene comprises a primer and a probe for the reference gene.

In some embodiments of the present invention, the detection reagent for the reference gene comprises a nucleotide sequence as set forth in SEQ ID NO: 22. SEQ ID NO:23, as shown in nucleotide sequence SEQ ID NO: a probe shown at 24.

In some embodiments of the present invention, the detection sample of the reagent is selected from at least one of saliva, sputum, nasopharyngeal aspirate, nasal swab, oropharyngeal swab, and nasopharyngeal swab.

In a second aspect of the present invention, a kit is provided, which contains the above-mentioned detection reagent.

In some embodiments of the invention, the kit further comprises a positive control and a negative control.

In some embodiments of the invention, the positive control comprises pseudo-viral particles of 2019-nCoV, IAV and IBV.

In some embodiments of the invention, the negative control is a pseudoviral particle containing an internal standard target fragment.

In some embodiments of the invention, the kit further comprises paraffin oil, a buffer, dNTPs, and an enzyme.

In some embodiments of the invention, the buffer is a PCR reaction buffer.

In some embodiments of the invention, the PCR reaction buffer comprises 40-60mM Tris solution, 70-80mM K ⁺ And 2-3mMMg ²⁺ 。

In some embodiments of the invention, the enzyme comprises HotStart Taq DNA polymerase and M-MLV reverse transcriptase.

In a third aspect of the invention, the application of the reagent or the kit is provided, and the application is the application in the preparation of novel coronavirus, influenza A virus and/or influenza B virus detection products.

In some embodiments of the invention, the detecting comprises the steps of: and detecting a sample to be detected by using the reagent or the kit.

In some embodiments of the present invention, the N gene or ORF1ab gene of the novel coronavirus to which the reagent or the kit is directed, the detection region of influenza a virus is the M1 gene, and the detection region of influenza b virus is the NEP gene.

In some embodiments of the present invention, the detection region of the novel coronavirus targeted by the reagent or the kit is a region as shown in SEQ id no:27 or SEQ ID NO:28, and the detection region of the influenza a virus is shown as SEQ ID NO:25, and the detection region of the influenza B virus is a sequence shown as SEQ ID NO:26, or a sequence shown in fig. 26.

In some embodiments of the invention, the detection is performed using a real-time fluorescent quantitative PCR reaction.

In some embodiments of the present invention, the amplification procedure of the real-time fluorescent quantitative PCR reaction is:

90-97℃10-40s

90-97℃0.5-5s 30-50cycles

56-64 deg.c (collecting fluorescence) 10-30s 30-50cycles.

92℃20s

92℃1s 40cycles

20s 40cycles at 60 deg.C (fluorescence collection).

In some embodiments of the present invention, the real-time fluorescent quantitative PCR reaction system is:

in some embodiments of the invention, when internal standard nucleic acids are detected using a FAM channel selected for detection of novel coronavirus nucleic acids, a Texas Red channel selected for detection of influenza a virus nucleic acids, a VIC channel selected for detection of influenza b virus nucleic acids, a CY5 channel selected, the criteria are:

(1) When the CT detected by the FAM channel is less than or equal to 36, the coronavirus is judged to be positive, and when the CT detected by the FAM channel is more than 36, the coronavirus is judged to be negative;

(2) When the CT detected by the Texas Red channel is less than or equal to 36, the influenza A virus is judged to be positive, and when the CT detected by the Texas Red channel is more than 36, the influenza A virus is judged to be negative;

(3) When the CT detected by the VIC channel is less than or equal to 36, the influenza B virus is judged to be positive, and when the CT detected by the VIC channel is more than 36, the influenza A virus is judged to be negative.

According to some embodiments of the invention, at least the following benefits are achieved: the reagent for rapidly detecting the influenza A virus, the influenza B virus and the novel coronavirus has high sensitivity, strong specificity and short detection time, does not have cross reaction with various other common respiratory pathogens, and has higher reference value for auxiliary diagnosis and clinical medication guidance of common influenza and the novel coronavirus. Meanwhile, the invention also overcomes the defect that the nucleic acid detection kit needs refrigeration or frozen transportation and storage by adopting the fluorescence detection and freeze-drying technology, can be transported at normal temperature, saves the cost and has long storage time; the primer probe group has high specificity and sensitivity for simultaneously detecting nucleic acids of novel coronavirus, influenza A virus and influenza B virus; the kit prepared by the primer probe group has the advantages of quick operation, simple method, good detection specificity, high sensitivity and multiple detections in one tube, and thoroughly solves the problems of low detection efficiency, poor specificity and low sensitivity of the kit in the prior art; the freeze-drying reaction mixing system disclosed by the invention does not need liquid preparation, can be directly used, shortens the pretreatment time, and has the same effect as a liquid reaction mixing system. The freeze-drying detection kit overcomes the defect that the nucleic acid detection kit needs refrigeration or frozen transportation for storage, can be transported at normal temperature, saves the cost, and further reduces the risk of aerosol pollution in the sample adding process.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a diagram showing the operation of example 4 of the present invention;

FIG. 2 is a graph showing the results of sensitivity detection of a novel coronavirus sample in example 5 of the present invention, wherein 1 is 4.0X 10 ⁵ copies/mL;2 is 4.0X 10 ⁴ copies/mL;3 is 4.0X 10 ³ copies/mL;4 is 4.0X 10 ² copies/mL;5 is a negative sample;

FIG. 3 is a graph showing the results of the sensitivity detection of seasonal H1N1 samples in example 5 of the present invention, wherein 1 is 1.5X 10 ³ TCID ₅₀ Per mL;2 is 1.5X 10 ² TCID ₅₀ Per mL;3 is 1.5X 10 ¹ TCID ₅₀ Per mL;4 is 1.5X 10 ⁰ TCID ₅₀ Per mL;5 is a negative sample;

FIG. 4 is a graph showing the results of the sensitivity test on the B/Victoria type sample in example 5 of the present invention, wherein 1 is 2.0X 10 ³ TCID ₅₀ Per mL;2 is 2.0X 10 ² TCID ₅₀ Per mL;3 is 2.0X 10 ¹ TCID ₅₀ Per mL;4 is 2.0X 10 ⁰ TCID ₅₀ Per mL;5 is a negative sample;

FIG. 5 is a graph showing the results of testing different types of novel coronavirus, influenza A virus and influenza B virus samples in example 6 of the present invention;

FIG. 6 is a graph showing the results of nucleic acid detection kits (fluorescence PCR) for the Senxiang Biotechnology, inc. of 2019-nCoV, influenza A virus and influenza B virus for different types of novel coronavirus, influenza A virus and influenza B virus samples according to example 6 of the present invention;

FIG. 7 is a graph showing the results of the measurement of the samples of the novel coronavirus, the influenza A virus and the influenza B virus on the HC800 instrument in example 7 of the present invention, wherein A is the result of the measurement of the sample of the novel coronavirus; b is the detection result of the influenza A virus sample; c is the detection result of the influenza B virus sample;

FIG. 8 is a graph showing the results of testing samples of the novel coronavirus, influenza A virus and influenza B virus on an ABI7500 apparatus in example 7 of the present invention, wherein A is the test result of the novel coronavirus sample; b is the detection result of the influenza A virus sample; and C is the detection result of the influenza B virus sample.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present invention belong to the scope of the freezing protection of the present invention.

Example 1 design and optimization of specific primer probes

This example optimizes specific primer probes designed for coronavirus, influenza a virus, and influenza b virus nucleic acids.

1. Primer design

In this example, through a large number of studies, a detection target region of a novel coronavirus (2019-nCoV) is located at an N gene and an ORF1ab gene, a detection target region of an influenza a virus is located at an M1 gene, a detection target region of an influenza b virus is located at an NEP gene, the above-mentioned target regions are used as templates, primer design is performed, in order to obtain a multiplex primer probe system combination with good sensitivity and specificity, a primer sequence and a probe sequence shown in table 1 are obtained by screening, and a multiplex test is combined. And human housekeeping gene beta-Globin is used as an internal standard quality control, a pair of primers (SEQ ID NO: 10-11) for detecting an internal standard sequence and a probe Taqman probe (SEQ ID NO: 12) are designed, 5 'and 3' of the probes are respectively marked with CY5 and BHQ2, the extraction and amplification processes of a target sequence are monitored, and false negative is avoided.

TABLE 1

/>

2. Primer Probe sensitivity test

In order to carry out more reasonable evaluation on the specificity effect of the designed primer probe and avoid the influence of multiple competition, a single-fluorescence PCR system and a multiple-fluorescence PCR system are respectively adopted to carry out a primer probe sensitivity test.

(1) Primer probe sensitivity test by single fluorescent PCR system

Extracting nucleic acid from novel coronavirus pseudovirus, influenza A virus H3N2 and influenza B virus BV samples (samples from Guangzhou Ruidan Biotech limited and Guangzhou respiratory disease research institute) by using a nucleic acid sample releasing agent (record number: guangdong and Xin health science and technology Limited 20210063), and diluting the nucleic acid into L1-L4 by 10 times by using a negative matrix (sample preserving fluid containing a negative throat swab); and synchronously extracting a negative control and a positive control.

Table 2 primer probe grouping was as follows:

the primer probe sequences shown in the table 1 are adopted, the primer probes shown in the table 2 are grouped, the real-time fluorescence RT-PCR is adopted to respectively detect the nucleic acids of the novel coronavirus, the influenza A virus and the influenza B virus samples with different concentration gradients, and an RT-PCR amplification system is as follows: probe 0.3. Mu.M, upstream and downstream primers 0.08. Mu.M, sample RNA to be tested 5. Mu.L (about 20 ng), 0.8mdNTPs, hotStart DNA polymerase 1.5U, M-MLV reverse transcriptase 40U, 1 XPCR reaction buffer, and enzyme-free sterile water to 20. Mu.L. Amplification conditions: pre-denaturation at 92 ℃ for 20s; finally, amplification was carried out at 95 ℃ for 1sec and 60 ℃ for 20sec for 40cycles, and fluorescence signal detection was carried out at the end of extension of each cycle.

TABLE 3 influenza A Virus Simplex sensitivity test results

TABLE 4 influenza B Virus singleplex sensitivity test results

TABLE 5 Single sensitivity test results for novel coronaviruses

The results are shown in tables 3-5, which show that the IAV primer probe set 1 is most sensitive and IBV primer probe set 1 is most sensitive.

(2) Primer probe sensitivity test by using 4-fold fluorescence PCR system and single-fold fluorescence PCR system

A mixed sample of influenza A virus, influenza B virus culture and novel coronavirus 2019-nCoV pseudovirus is used as a template, a negative matrix is diluted by 10 times to form L1-L4 gradient dilution, a primer probe set sequence adopted by an IAV single-fold fluorescent quantitative PCR experiment is shown as SEQ ID NO:1-3, a primer probe set sequence adopted by an IBV single-fold reagent fluorescent quantitative PCR experiment is shown as SEQ ID NO:4-6, a primer probe set sequence adopted by a 2019-nCoV single-fold reagent fluorescent quantitative PCR experiment is shown as SEQ ID NO:7-9, a primer probe set sequence adopted by an internal standard single-fold reagent fluorescent quantitative PCR experiment is shown as SEQ ID NO:10-12, a primer probe set sequence adopted by a quadruple reagent fluorescent quantitative PCR experiment is shown as SEQ ID NO:1-12, and a detection system and amplification conditions are consistent with those of a single-fold fluorescent PCR system for primer sensitivity test.

TABLE 6 verification of sensitivity of influenza A Virus

The results are shown in table 6, and it can be seen from table 6 that 4-fold PCR detection has no influence on the results, and in conclusion, the sequences of the primer probe combinations for the novel coronavirus, influenza a virus and influenza b virus with high sensitivity are finally selected and selected as shown in table 7 below.

TABLE 7

Numbering	Group of groups	Primer sequences
			SEQ ID NO:1	IAV-F	5’-GAGGTCGAAACGTAYGTTCTYTCTATC-3’
SEQ ID NO:2	IAV-R1	5’-CATCTTCYAGTCTCTGYGCGAT-3’
			SEQ ID NO:3	IAV-P-TXR	5’-CGTCAGGCCCCCTCAAAGCCG-3’
SEQ ID NO:4	IBV-F	5’-AGTACTCTATGGTTGGGTATGAAGC-3’
			SEQ ID NO:5	IBV-R	5’-TTGCATCATCTCCCATTCTTAATA-3’
SEQ ID NO:6	IBV-P-FAM	5’-TGGCTCTTTACAATATGGCAACACCTGTGTC-3’
			SEQ ID NO:7	CDCN-F	5’-GGGGAACTTCTCCTGCTAGAAT-3’
SEQ ID NO:8	CDCN-R	5’-CAGACATTTTGCTCTCAAGCTG-3’
			SEQ ID NO:9	CDCN-P-FAM	5’-TTGCTGCTGCTTGACAGATT-3’
SEQ ID NO:10	ORF1ab-mF	5’-GGTTTGAAGTCCATCTCCTAAGC-3’
			SEQ ID NO:11	ORF2ab-mR	5’-CACAGGGTGAGGTGTAAGTGAT-3’
SEQ ID NO:12	CDCFZ-P-VIC	5’-ACCTGTCCTTGGCTCTTCTGGCACT-3’

Aiming at the optimal primer probe combination shown in Table 7, the optimal detection target region for detecting the influenza A virus is NC-002016.114-116 bp (shown as SEQ ID NO. 25), the optimal detection target region for detecting the influenza B virus is AF100359.1 944-1135 bp (shown as SEQ ID NO. 26), the optimal detection target region for detecting the novel coronavirus N gene is MN908947.328880-28978bp (shown as SEQ ID NO. 27), the optimal detection target region for detecting the novel coronavirus ORF1a gene is MN 6253 zxft 6213330-13462 bp (shown as SEQ ID NO. 28), and the optimal detection target region for detecting the internal reference gene beta-Globin is MKK959.135bp-114 bp (shown as SEQ ID NO. 29). The sequence of the target region is shown in table 8.

TABLE 8

Example 2 novel detection kit for coronavirus, influenza A virus and influenza B virus

This example prepared a novel assay kit for coronavirus, influenza a virus and influenza b virus, comprising a lyophilization reaction 8 manifold of a 2019-nCoV/IAV/IBV lyophilization reaction mixture, a 2019-nCoV/IAV/IBV positive control (lyophilized powder), a negative control (lyophilized powder), paraffin oil, which was a mixture of the two reactionsIn the 8-joint freeze-drying reaction of the 2019-nCoV/IAV/IBV freeze-drying reaction mixture, the main components comprise (1) freeze-dried powder of a primer and a probe set for detecting target sequence amplification (a primer and probe combination (the sequence is shown in Table 7) of the novel coronavirus, the influenza A virus and/or the influenza B virus screened in example 1); (2) PCR buffer freeze-dried powder (50 mM Tris solution, 75mM K) ⁺ 、2.4mMMg ²⁺ ) (ii) a (3) 25mM dNTPs freeze-dried powder; (4) enzyme mixture freeze-dried powder: hotStart Taq DNA polymerase and M-MLV reverse transcriptase; and (5) freeze-drying the freeze-drying preservation solution. Wherein, the ratio of each component in the lyophilization reaction 8 union of the 2019-nCoV/IAV/IBV lyophilization reaction mixture is shown in Table 9.

TABLE 9

Grouping of groups	Final concentration
		Freeze-drying freezing-protecting liquid	50％
Buffer solution for PCR reaction	1X (50 mM Tris solution, 75mM K) ⁺ 、2.4mMMg ²⁺ )
		dNTPs	0.8mM
SEQ ID NO:1	0.08μM
		SEQ ID NO:2	0.08μM
SEQ ID NO:3	0.3μM
		SEQ ID NO:4	0.06μM
SEQ ID NO:5	0.06μM
		SEQ ID NO:6	0.3μM
SEQ ID NO:7	0.1μM
		SEQ ID NO:8	0.1μM
SEQ ID NO:9	0.4μM
		SEQ ID NO:10	0.05μM
SEQ ID NO:11	0.05μM
		SEQ ID NO:12	0.2μM
HotStart DNA polymerase	1.5U
		M-MLV reverse transcriptase	40U

Example 3 establishment of fast detection method

The kit in the embodiment 2 of the invention is used for establishing a method for detecting a sample.

1. The detection method of the invention is real-time fluorescence RT-PCR.

By screening various mutant Taq DNA polymerases, the amplification time is shortened and the adverse resistance is realized. 2kb can be amplified in 1 minute, namely the amplification product of 100bP can complete the amplification of 100bP in 3 seconds, while the conventional taq enzyme can amplify only 1kb in 1 minute. Therefore, the extension time in the amplification procedure can be greatly shortened compared to conventional reagents, making the amplification procedure shorter. Based on this, the optimum reaction conditions were clarified as follows.

Reverse transcription: 48 ℃ for 300s

2. Fluorescence detection channel selection:

(1) Selecting a FAM channel for detection of novel coronavirus nucleic acids;

(2) Selecting a Texas Red channel to detect influenza A virus nucleic acid;

(3) Selecting VIC channel to detect influenza B virus nucleic acid;

(4) The CY5 channel is selected and the internal standard nucleic acid is detected.

3. Analysis of results

And (3) carrying out reaction by using an UltraFast QPCR real-time fluorescence quantitative PCR instrument, automatically storing a result after the reaction is finished, recording a Ct value of the sample automatically analyzed and calculated by the instrument, and directly printing a detection result.

(1) Negative and positive controls are required for each test batch for quality control. The following requirements need to be met simultaneously in the same experiment, otherwise, the experiment is invalid.

Negative control: fluorescence signals of fluorescence channels of FAM, texas Red and VIC are not obviously increased and have no obvious S type;

an amplification curve; the fluorescence signal in CY5 fluorescence channel has obvious increase, which presents a typical S-shaped amplification curve, and Ct value is less than or equal to 36.0.

Positive control: fluorescence signals of fluorescence channels of FAM, texas Red and VIC are obviously increased and are in typical S-shaped curves, and the Ct value is less than or equal to 36.00; the Ct value in the CY5 fluorescence channel is not required.

(2) Results were analyzed as per table 10 with all positive, negative and internal control results normal:

watch 10

Note: "+" indicates a positive result, and "-" indicates a negative result; "NAN" means no amplification.

EXAMPLE 4 method of Using the kit

The invention uses nucleic acid sample releasing agent (record number: yuejiu equipment 20210063) produced by Guangdong and Xin health science and technology Limited company to extract nucleic acid from different types of novel coronavirus, influenza A virus and influenza B virus samples, and synchronously extracts negative control and positive control.

The specific operation method of the invention is as follows:

(1) Placing the two freeze-drying reaction 8-union tube pairs containing the 2019-nCoV/IAV/IBV freeze-drying reaction mixture into a palm centrifuge (paying attention to balance), centrifuging for 3 seconds, and throwing freeze-drying powder in the tube to the bottom of the tube so as to prevent the freeze-drying powder from flying out when a cover is opened;

(2) Opening the 8-tube cover, and vertically dripping 1 drop (about 20 mu L) of nucleic acid of a sample to be detected into the first hole;

(3) One drop (about 20. Mu.L) of reconstituted negative control was added vertically drop-wise to the second well;

(4) One drop (about 20 mu L) of redissolved positive control is vertically dropped into the third hole (when the solution is added, a dropper is not needed to be touched);

(5) Dripping 1 drop of paraffin oil (when adding liquid, the dropper is not touched), covering 8 tubes with a cover, turning upside down for 5 times, mixing uniformly, placing the 8 tubes in a palm centrifuge, centrifuging for 10 seconds to ensure that the paraffin oil is completely on the upper layer of the reaction mixed liquid (if bubbles exist in the tubes, the tube walls are flicked to remove the bubbles, and then centrifuging again);

(6) PCR reaction was performed.

The operation method of the invention is shown in figure 1, the freeze-drying reaction mixed system of the invention does not need liquid preparation, can be directly used, and is convenient and rapid. Example 5 Performance test of the kit of the present invention

1. Test for accuracy of detection

(1) Nucleic acid extraction

Nucleic acid extraction was performed on novel coronavirus pseudoviruses, influenza a virus H3N2 and influenza b virus BV samples (samples from guangzhou ruida biotechnology limited and guangzhou respiratory disease institute)) using a nucleic acid sample releasing agent (record No.: yue ear instrument No. 20210063) produced by guangdong and health science limited, and a negative control and a positive control were simultaneously extracted.

(2) Nucleic acid detection

mu.L of the sample nucleic acid obtained by the above extraction was added to a lyophilization reaction 8-line of the mixture of the novel coronavirus, influenza A virus and influenza B virus lyophilization reactions and assayed according to the assay method described in example 3.

(3) Analysis of results

By using the kit prepared in example 1 and the detection method of example 3 to detect samples of different types of novel coronaviruses (2019-nCoV), influenza A Virus (IAV) and Influenza B Virus (IBV), the test results of the detection accuracy of the virus samples are shown in table 11, which indicates that the kit of the present invention can specifically detect: influenza a virus seasonal H1N1, seasonal H3N2, H7N9, new H1N1; influenza b viruses Yamagata type, victoria type; novel coronavirus pseudovirions, novel coronavirus positive sample nucleic acids.

TABLE 11

Note: "NAN" means no amplification.

2. Cross-reaction experiments with other pathogens

(1) Nucleic acid extraction

The nucleic acid sample release agent (record No.: yuejie Instrument No. 20210063) produced by Guangdong and Xin health science Co., ltd.) was used to treat rubella virus (1X 10) ⁵ TCID ₅₀ /mL), mumps virus (1X 10) ⁵ TCID ₅₀ /mL), measles virus (1X 10) ⁵ TCID 50/mL), staphylococcus aureus (1X 10) ⁶ CFU/mL), escherichia coli (1X 10) ⁶ CFU/mL), pseudomonas aeruginosa (1X 10) ⁶ CFU/mL), enterovirus 71 (1X 10) ⁵ TCID ₅₀ PermL), coxsackie virus type A16 (1X 10) ⁵ TCID ₅₀ mL), respiratory syncytial virus type A (1X 10) ⁵ TCID ₅₀ mL), respiratory syncytial virus type B (1X 10) ⁵ TCID ₅₀ /mL), parainfluenza virus type 1 (1X 10) ⁵ TCID ₅₀ mL), parainfluenza virus type 2 (1X 10) ⁵ TCID 50/mL), parainfluenza virus type 3 (1X 10) ⁵ TCID ₅₀ Perml), cytomegalovirus (1X 10) ⁵ TCID 50/mL), mycoplasma pneumoniae (1X 10) ⁵ TCID ₅₀ mL), chlamydia pneumoniae (1X 10) ⁶ CCU/mL), haemophilus influenzae (1X 10) ⁶ CFU/mL), neisseria meningitidis (1X 10) ⁶ CFU/mL), lactobacillus casei (1X 10) ⁶ CFU/mL), streptococcus pneumoniae (1X 10) ⁶ CFU/mL), salmonella enteritidis (1X 10) ⁶ CFU/mL), beta hemolytic streptococcus (1X 10) ⁶ CFU/mL), proteus vulgaris (1X 10) ⁶ CFU/mL), EB virus (1X 10) ⁵ TCID ₅₀ mL) sample, and extracting the nucleic acid, and synchronously extracting a negative control and a positive control.

(3) Nucleic acid detection

mu.L of the sample nucleic acid obtained by the above extraction was added to a lyophilization reaction 8-line of the mixture of the novel coronavirus, influenza A virus and influenza B virus lyophilization reactions and assayed according to the assay method described in example 4.

(4) Analysis of results

By using the kit prepared in example 1 and the detection method of example 4, rubella virus, mumps virus, measles virus, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, enterovirus 71, coxsackievirus a16, respiratory syncytial virus a, respiratory syncytial virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, cytomegalovirus, mycoplasma pneumoniae, chlamydia pneumoniae, haemophilus influenzae, neisseria meningitidis, lactobacillus casei, streptococcus pneumoniae, salmonella enteritidis, streptococcus B hemolyticus, proteus vulgaris, EB virus and other pathogens are detected, specific data of detection results are shown in table 12, and results show that the kit has no cross reaction with the pathogens, thereby showing that the kit has high specificity.

TABLE 12

Pathogens	The result of the detection	Pathogens	The result of the detection
				Rubella virus	Negative of	Mycoplasma pneumoniae	Negative of
Mumps virus	Negative of	Chlamydia pneumoniae	Negative of
				Measles virus	Negative of	Parainfluenza virus type 1	Negative of
Staphylococcus aureus (Staphylococcus aureus)	Negative of	Parainfluenza virus type 2	Negative of
				Escherichia coli	Negative of	Parainfluenza virus type 3	Negative of
Pseudomonas aeruginosa	Negative of	Neisseria meningitidis	Negative of
				Enterovirus 71	Negative of	Lactobacillus casei	Negative of
Coxsackievirus A16 type	Negative of	Streptococcus pneumoniae	Negative of
				Respiratory syncytial virus type A	Negative of	Salmonella enteritidis	Negative of
Respiratory syncytial virus type B	Negative of	Beta hemolytic streptococcus	Negative of
				Haemophilus influenzae	Negative of	Proteus vulgaris	Negative of
Cytomegalovirus	Negative of	EB virus	Negative of

3. Sensitivity test

(1) Nucleic acid extraction

Nucleic acid sample release agents (record No.: yueji Instrument No. 20210063) from Guangdong and Xin health science Co., ltd.) were used for 4.0X 10 of the novel coronavirus (pseudovirus particle) respectively ² ～4.0×10 ⁵ Samples of copies/mL, influenza A virus (seasonal H1N 1) 1.5X 10 ⁰ ～1.5×10 ³ TCID ₅₀ PermL sample, influenza B virus (B/Victoria) 2.0X 10 ⁰ ～2.0×10 ³ TCID ₅₀ The nucleic acid extraction was performed per mL of sample.

(3) Nucleic acid detection

(4) Analysis of results

Novel coronavirus (pseudovirion) 4.0X 10 by using the kit prepared in example 1 and the detection method in example 3 ² ～4.0×10 ⁵ Samples of copies/mL, influenza A virus (seasonal H1N 1) 1.5X 10 ⁰ ～1.5×10 ³ TCID ₅₀ Perml sample, influenza B virus (B)/Victoria)2.0×10 ⁰ ～2.0×10 ³ TCID ₅₀ The novel coronavirus detection results are shown in FIG. 2 (curves No.1, 2, 3 and 4 are respectively 4.0 × 10 of the novel coronavirus) ⁵ copies/mL、4.0×10 ⁴ copies/mL、4.0×10 ³ copies/mL、4.0×10 ² The samples were copies/mL and curve 5 was a negative sample), and the results of detection of influenza A virus are shown in FIG. 3 (curves 1, 2, 3 and 4 are 1.5X 10 of seasonal H1N1, respectively ³ TCID ₅₀ /mL、1.5×10 ² TCID ₅₀ /mL、1.5×10 ¹ TCID ₅₀ /mL、1.5×10 ⁰ TCID ₅₀ The sample was mL, curve 5 was a negative sample), and the results of detecting influenza B virus are shown in FIG. 4 (curves 1, 2, 3 and 4 were 2.0X 10 of B/Victoria type, respectively ³ TCID ₅₀ /mL、2.0×10 ² TCID ₅₀ /mL、2.0×10 ¹ TCID ₅₀ /mL、2.0×10 ⁰ TCID ₅₀ a/mL sample, curve 5 is a negative sample), which shows that the kit can detect 4.0 multiplied by 10 ² Samples of novel coronavirus pseudovirions at concentrations above copies/mL, 1.5X 10 ⁰ TCID ₅₀ Samples of seasonal H1N1 at concentrations above/mL and 2.0X 10 ⁰ TCID ₅₀ The B/Victoria type sample with the concentration higher than/mL indicates that the kit has higher sensitivity, the specific data are shown in Table 13, and the Table 13 is a sensitivity experiment result table.

Watch 13

Note: "NAN" means no amplification.

4. Precision experiment

The kit prepared in example 1 was used to test the precision reference QJ1 (N gene 16000copies/mL, M1 gene 64 TCID) ₅₀ mL, NEP Gene 80TCID ₅₀ mL) and QJ2 (N gene 1000copies/mL, M1 gene 4TCID ₅₀ mL, NEP Gene 5TCID ₅₀ mL) and repeating the detection 10 times to calculate ORF1ab gene, N gene, M1 groupCoefficient of variation CV% due to the sum of Ct values of NEP gene.

Precision reference products QJ1 and QJ2 were tested 10 times in duplicate using novel coronavirus (2019-nCoV), influenza A Virus (IAV) and Influenza B Virus (IBV) nucleic acid assays, and the results are shown in table 14. The Ct value variation coefficients of the N gene, the M1 gene and the NEP gene for the low-concentration precision reference product QJ1 are respectively 1.14%, 1.43% and 1.07%, and the Ct value variation coefficients for the low-concentration precision reference product QJ2 are respectively 1.48%, 1.49% and 1.31%, and are all less than 5%. The freeze-drying kit for detecting the nucleic acid of the novel coronavirus (2019-nCoV), the Influenza A Virus (IAV) and the Influenza B Virus (IBV) constructed by the research has good repeatability.

TABLE 14

/>

Example 6 comparison of the detection results of the method of the present invention and nucleic acid detection kit (fluorescence PCR method) for detecting novel coronavirus 2019-nCoV, influenza A virus and influenza B virus of Santa Clara Biotechnology Ltd

(1) Preparation of nucleic acid amplification reaction liquid for novel coronavirus, influenza A virus and influenza B virus

The nucleic acid amplification reaction solution of the novel Saint Xiang coronavirus, the novel influenza A virus and the novel influenza B virus is prepared by a nucleic acid detection kit method of the novel Saint Xiang coronavirus 2019-nCoV, the novel influenza A virus and the novel influenza B virus.

The product does not need to prepare an amplification reaction solution.

(2) Nucleic acid extraction

The invention uses nucleic acid sample releasing agent (record number: yueji mechanical preparation 20210063) produced by Guangdong and Xin health science and technology Limited company to extract nucleic acid from novel coronavirus, influenza A virus and influenza B virus samples of different types, and synchronously extracts negative control and positive control; meanwhile, nucleic acid extraction or purification reagents (Xiangchan Shijie Ning Yuan Shigao 20150021) of Santa Clariti Biotechnology corporation are adopted to carry out nucleic acid extraction on novel coronavirus, influenza A virus and influenza B virus samples of different types, negative control and positive control according to the relevant operations of the instruction manual.

(3) Nucleic acid detection

Adding 20 μ L of the sample nucleic acid obtained by the above step into 8 connecting tubes of freeze-drying reaction of the freeze-drying reaction mixture of the novel coronavirus, influenza A virus and influenza B virus, and detecting according to the detection method in example 3; meanwhile, nucleic acid detection kits (fluorescence PCR method) for detecting novel coronavirus 2019-nCoV, influenza A virus and influenza B virus of Shengxiang biological science and technology Co., ltd are adopted for detection, the reagent preparation method is shown in the following table 15, and the reagent preparation method for nucleic acid detection kits (fluorescence PCR method) for novel coronavirus 2019-nCoV, influenza A virus and influenza B virus of Shengxiang biological science and technology Co., ltd is shown in the table 15.

Watch 15

The amplification reaction procedure with nucleic acid detection kit (fluorescence PCR method) for novel coronavirus 2019-nCoV, influenza A virus and influenza B virus of Shengxiang Biotechnology Ltd is as follows:

(4) Analysis of results

The result shows that the detection result of the method has no obvious difference from the detection result of the kit of Shangxiang biotechnology, inc. However, the invention does not need to prepare liquid, and the extraction process is simpler; the method needs shorter time in the nucleic acid detection process, only needs about 30min, and needs about 60min for the Shengxiang reagent, so the method can better save the time. Specific detection results are shown in fig. 5-6 and table 16 (fig. 5 is a test result of the invention, fig. 6 is a test result of the saint xiang biotechnology company, ltd. Kit), and table 12 is a comparison table of the detection results of the method of the invention and the saint xiang biotechnology company, ltd. Kit, and the results show that the kit prepared by the scheme of the invention has better detection sensitivity compared with the saint xiang biological kit.

TABLE 16

Note: "NAN" and "Undet" indicate no amplification.

Example 7 comparison of the detection effects of different detection instruments according to the method of the present invention

(1) Nucleic acid extraction

The invention uses nucleic acid sample releasing agent (record number: yuejiu equipment 20210063) produced by Guangdong and Xin health science and technology Limited company to extract nucleic acid from novel coronavirus, influenza A virus and influenza B virus samples, and synchronously extracts negative control and positive control.

(3) Nucleic acid detection

Adding 20 μ L of the sample nucleic acid obtained by the above extraction into a freeze-drying reaction 8-tube connector of a freeze-drying reaction mixture of the novel coronavirus, the influenza A virus and the influenza B virus, and detecting according to the detection method in example 3; the detection is carried out by using two different detection instruments, namely HC800 and ABI 7500.

(5) Analysis of results

The result shows that the detection result of the method has no obvious difference from the detection result of the kit of Shangxiang biotechnology, inc. However, the invention does not need to prepare liquid, and the extraction process is simpler; the method needs shorter time, only 32min, and can save time well in the nucleic acid detection process. The specific test results are shown in fig. 7-8 and table 17 (fig. 7 is the HC800 test result, fig. 8 is the ABI7500 test result), and table 17 is a comparison table of the test effects of different test instruments according to the method of the present invention.

TABLE 17

Note: "NAN" and "Undet" indicate no amplification.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. A reagent for detecting a novel coronavirus, influenza a and/or influenza b virus nucleic acid, wherein the reagent comprises a first primer combination for detecting influenza a virus, a second primer combination for detecting influenza b virus, and a third primer combination for detecting novel coronavirus;

the third primer combination comprises a primer group with a sequence shown in SEQ ID NO.7 and SEQ ID NO.8 or a primer group with a sequence shown in SEQ ID NO.22 and SEQ ID NO. 23.

2. The reagent of claim 1, further comprising a probe sequence; preferably, the probe sequence is selected from one or more of SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 15, SEQ ID NO 18, SEQ ID NO 21 and SEQ ID NO 24.

3. The reagent of claim 2, wherein the 5' end of the probe comprises a fluorescence reporter group and a fluorescence quencher group; preferably, the fluorescence reporter group comprises any one of FAM, texas Red, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY5 and VIC, and the fluorescence quencher group comprises any one of MGB, BHQ-1, BHQ-2 and BHQ-3.

4. The reagent according to claim 1, wherein the reagent further comprises a detection reagent for an internal reference gene; preferably, the reference gene is beta-Globin or GAPDH; more preferably, the detection reagent for the reference gene comprises a primer and a probe aiming at the reference gene.

5. The reagent according to claim 1, wherein the test sample further comprises at least one selected from the group consisting of saliva, sputum, nasopharyngeal aspirate, nasal swab, oropharyngeal swab, and nasopharyngeal swab.

6. A kit comprising the detection reagent according to any one of claims 1 to 5.

7. The kit of claim 6, further comprising positive and negative controls, paraffin oil, buffers, dNTPs, and enzymes; preferably, the enzymes include HotStart Taq DNA polymerase and M-MLV reverse transcriptase.

8. Use of the reagent according to any one of claims 1 to 5 or the kit according to claim 6 or 7 for the preparation of novel coronavirus, influenza a virus and/or influenza b virus detection products.

9. The use of claim 8, wherein the reagent or the kit is directed against N gene or ORF1ab gene of a novel coronavirus, the detection region of influenza A virus is M1 gene, and the detection region of influenza B virus is NEP gene.

10. Use according to claim 8, characterized in that said detection comprises the following steps: detecting nucleic acid in a sample to be tested using the reagent according to any one of claims 1 to 5 or the kit according to claim 6 or 7.