Nucleic acid composition for respiratory tract pathogen detection and application thereof

Abstract

The invention provides a nucleic acid composition for respiratory tract pathogen detection and application thereof, and relates to the technical field of biology. The invention provides a nucleic acid detection composition which has high specificity and can be used for detecting various respiratory pathogens. The composition can be used for detecting influenza B virus, influenza A virus H1N1, influenza A virus H3N2, novel coronavirus N gene, novel coronavirus O gene, respiratory syncytial virus, I-type parainfluenza virus, II-type parainfluenza virus, III-type parainfluenza virus, IV-type parainfluenza virus, rhinovirus, adenovirus, haemophilus influenzae, and the like. The kit has 15 pathogens including streptococcus pneumoniae and staphylococcus aureus, the lower detection limit can be as low as 20 copies / reaction, and the kit plays an important role in rapid detection of respiratory tract pathogens.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a Used for Nucleic acid compositions for respiratory pathogen detection and their applications. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Respiratory infections are infectious diseases caused by a variety of microorganisms including bacteria, viruses, mycoplasmas, fungi, parasites, etc. About 90% of respiratory infections are caused by viruses, and bacterial infections often follow viral infections. Respiratory infectious diseases pose a huge threat to human health and social activities, and examples of large-scale epidemics caused by them are also common. The rapid spread and easy mutation of respiratory diseases have greatly increased the difficulty of prevention and control. Respiratory infectious diseases are mainly infectious diseases caused by pathogens invading the patient's body through the nasal cavity, trachea, throat and bronchi. The pathogens include various bacteria, viruses, mycoplasmas and chlamydia, etc., which are mainly transmitted through air, droplets and contact.

[0004] There are many common methods for detecting respiratory pathogens, including nucleic acid testing, antigen / antibody testing, microbial culture, serological testing, etc. Nucleic acid testing has high sensitivity and good specificity. It can accurately distinguish different pathogens even when there is very little pathogen nucleic acid in the sample, so it has a wide range of applications. Some existing nucleic acid respiratory pathogen detection products have fewer detection items. For example, CN119020535A can only detect 12 common respiratory pathogens, which cannot meet market demand well. In addition, although some can detect more common respiratory pathogens, they still have problems with insufficient specificity or sensitivity.

[0005] Based on the above situation, it is necessary to provide a nucleic acid composition with good specificity and sensitivity and capable of detecting multiple respiratory pathogens. Summary of the invention

[0006] The object of the present invention is to provide a nucleic acid composition for respiratory pathogen detection to alleviate the defects of respiratory pathogen detection in the prior art. Based on the above primer-probe composition, another object of the present invention is to provide the application of the above primer-probe composition and products using them.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] It should be noted that:

[0009] In the present invention, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution.

[0010] In the present invention, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0011] In the present invention, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution. In the present invention, unless otherwise specified, the numerical range "a-b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" have been listed in this article, and "6-22" is just an abbreviated representation of these numerical combinations.

[0012] The “range” disclosed in the present invention is in the form of a lower limit and an upper limit, which can be one or more lower limits, and one or more upper limits, respectively.

[0013] In the present invention, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance can but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur.

[0014] In the present invention, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but do not exclude any other elements, integers or steps.

[0015] In the present invention, the terms "each... is independently selected from" and "... are each independently selected from" and "... are independently selected from" are interchangeable and should be understood in a broad sense, which means that when selecting elements from a set, the selection of each element is independent of each other and is not affected by the selection of other elements.

[0016] In a first aspect, the present invention provides a nucleic acid composition for detecting respiratory pathogens, comprising at least one of nucleic acid compositions 1 to 15 for detecting influenza B virus, influenza A virus H1N1, influenza A virus H3N2, novel coronavirus N gene, novel coronavirus O gene, respiratory syncytial virus, parainfluenza virus type I, parainfluenza virus type II, parainfluenza virus type III, parainfluenza virus type IV, rhinovirus, adenovirus, Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus as shown in Table 1.

[0017] In an optional embodiment, a nucleic acid combination for detecting an internal reference gene is also included.

[0018] As a preferred embodiment of the present invention, the present invention provides a nucleic acid combination 16 for detecting a β-Actin internal reference gene.

[0019] Specifically, the nucleotide sequences contained in each nucleic acid combination 1 to 16 are shown in Table 1 below.

[0020] Table 1 Nucleic acid composition sequence

[0021]

[0022]

[0023]

[0024] *R is A or G; Y is C or T; M is A or C

[0025] Generally, taking nucleic acid combination 1 as an example, it includes a forward primer, a reverse primer and a probe, wherein the nucleotide ratio of each primer sequence is 1:1; similarly, nucleic acid combination 11 for detecting human rhinovirus is a nucleotide mixture comprising 3 forward primers and 1 reverse primer sequence. The ratio of each primer sequence is forward human rhinovirus primer 1: forward human rhinovirus primer 2: forward human rhinovirus primer 3 is 1:1:1:1. In the present invention, unless otherwise specified, the rest is the same.

[0026] In an optional embodiment, the nucleic acid composition of the present invention comprises at least one of the following (A) to (E):

[0027] (A) the nucleic acid combination 1 for detecting influenza B virus and the nucleic acid combination 12 for detecting adenovirus;

[0028] (B) the nucleic acid combination 2 for detecting influenza A virus H1N1 and the nucleic acid combination 14 for detecting Streptococcus pneumoniae;

[0029] (C) the nucleic acid combination 6 for detecting respiratory syncytial virus and the nucleic acid combination 13 for detecting Haemophilus influenzae;

[0030] (D) the nucleic acid combination 4 for detecting the novel coronavirus N gene, the nucleic acid combination 7 for detecting type I parainfluenza virus, the nucleic acid combination 8 for detecting respiratory type II parainfluenza virus, and the nucleic acid combination 10 for detecting respiratory type IV parainfluenza virus;

[0031] (E) The nucleic acid combination 5 for detecting the novel coronavirus O gene and the nucleic acid combination 15 for detecting Staphylococcus aureus.

[0032] In each nucleic acid combination, each probe is labeled with a luminescent group and a quenching group; wherein the luminescent group is labeled at the 5' end of the probe, and the quenching group is labeled at the 3' end of the probe.

[0033] In an optional embodiment, the luminescent group includes but is not limited to quantum dots, FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red or Cy5.

[0034] In an optional embodiment, the quenching group includes but is not limited to BHQ1, BHQ2, BHQ3 or MGB.

[0035] In an optional embodiment, the working concentration of the forward primer is 0.1-0.6 μM, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 μM, preferably 0.4 μM.

[0036] In an optional embodiment, the working concentration of the reverse primer is 0.1-0.6 μM, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 μM, preferably 0.4 μM.

[0037] In an optional embodiment, the working concentration of the probe is 0.05-0.4 μM, for example, but not limited to, 0.05, 0.1, 0.2, 0.3, or 0.4 μM, preferably 0.2 μM.

[0038] In a second aspect, there is provided a use of the nucleic acid composition for detecting human respiratory pathogens according to the first aspect in preparing a human respiratory pathogen detection product.

[0039] In a third aspect, a human respiratory pathogen detection kit is also provided, wherein the human respiratory pathogen detection kit comprises the nucleic acid composition for human respiratory pathogen detection described in the first aspect.

[0040] In an optional embodiment, the kit further comprises one or more of an enzyme mixture, a PCR reaction buffer, dNTPs, a negative control, a positive control and a blank control;

[0041] Optionally, the enzyme mixture includes reverse transcriptase, Taq enzyme, and UNG enzyme;

[0042] Optionally, the buffer comprises a buffer, magnesium ions and a protective agent.

[0043] The above substances for PCR reaction can be packaged separately or premixed in the same reagent, for example, premixed into a reaction buffer reagent. Each substance or reagent in the kit can be present in the form of a working reagent or a reserve reagent. When it is a reserve reagent, it is diluted to a working concentration before use.

[0044] In a fourth aspect, there is provided a primer-probe combination for detecting human respiratory pathogens as described in the first aspect, or a human respiratory pathogen detection kit as described in the third aspect for use in detecting human respiratory pathogens for non-diagnostic and therapeutic purposes.

[0045] In an optional embodiment, the application includes using the primer-probe combination for detecting human respiratory pathogens described in the first aspect, or the human respiratory pathogen detection kit described in the third aspect to perform PCR amplification on the sample to be tested.

[0046] In an optional embodiment, the PCR reaction procedure is:

[0047] 50°C, 2 min, 1 cycle;

[0048] 60°C, 20 min, 1 cycle;

[0049] 95°C, 1 min, 1 cycle;

[0050] 95°C for 10 sec, 58°C for 45 sec (collecting fluorescence), 45 cycles.

[0051] In an optional embodiment, the determination of the test results in the application can be based on mature and well-known methods in the art, such as judging whether the corresponding respiratory pathogen exists in the sample to be tested based on the appearance of an amplification curve, and a standard curve can be further constructed for quantitative detection, which is not limited by the present invention.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The present invention provides a highly specific nucleic acid detection composition that can be used to detect a variety of respiratory pathogens. The composition can detect influenza B virus, influenza A virus H1N1, influenza A virus H3N2, novel coronavirus N gene, novel coronavirus O gene, respiratory syncytial virus, parainfluenza virus type I, parainfluenza virus type II, parainfluenza virus type III, parainfluenza virus type IV, rhinovirus, adenovirus, Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus, a total of 15 pathogens, the detection limit can be as low as 20 copies / reaction, which plays an important role in the rapid detection of respiratory pathogens. DETAILED DESCRIPTION

[0054] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The enzyme mixture used in the following examples (consisting of reverse transcriptase, Taq enzyme, and UNG enzyme; reagents produced by Livzon).

[0056] Example 1 Detection of multiple primer probes on a digital microfluidic chip

[0057] The corresponding primers and probe sequences with corresponding fluorescent groups were synthesized according to Table 2.

[0058] Table 2 Nucleic acid composition sequence

[0059]

[0060]

[0061]

[0062]

[0063] *R is A or G; Y is C or T; M is A or C

[0064] Using the primers in Table 2, the reaction system was configured according to Tables 3 and 4. An average of about 5.5 μL of each reaction well was dispensed onto 8 sites on the digital microfluidic chip, and amplification detection was performed using a fully automatic digital microfluidic nucleic acid detector.

[0065] Table 3 PCR amplification system

[0066]

[0067] The samples to be tested are as follows:

[0068] Take each plasmid template except HPIV2 and HPIV4 in Table 4 and mix them to obtain 4 test samples containing 0, 20, 200, and 2000 copies of each plasmid template, respectively.

[0069] In addition, test samples containing 0, 20, 200, and 2000 copies of HPIV2 or HPIV4 plasmid templates were separately prepared.

[0070] Table 4 Plasmid template

[0071] Template Name Target Influenza B virus plasmid INFB Influenza A virus H1N1 plasmid H1N1 Influenza A virus H3N2 plasmid H3N2 Novel coronavirus N plasmid SARS-CoV-2(N) Novel Coronavirus Orf1ab Plasmid SARS-CoV-2(O) RSV A plasmid RSV Parainfluenza virus type 1 plasmid HPIV1 Parainfluenza virus type 2 plasmid HPIV2 Parainfluenza virus type 3 plasmid HPIV3 Parainfluenza virus type 4 plasmid HPIV4 Rhinovirus plasmid HRV Adenovirus plasmid HADV Haemophilus influenzae plasmid HI Streptococcus pneumoniae plasmid SP Staphylococcus aureus plasmid SA Human internal standard β-Actin

[0072] The reaction conditions are shown in Table 5.

[0073] Table 5 Reaction conditions

[0074]

[0075] The final test results are shown in Table 6 (ct value>40 is negative).

[0076] Table 6 Test results

[0077]

[0078] As can be seen from the results in Table 6, no amplification signal was observed in NTC, and there was no non-specific amplification between the primer-probe combination pairs of each combination; under the template concentration conditions of 2000 copies / reaction, 200 copies / reaction, and 20 copies / reaction, all plasmid templates containing the target gene sequence can be stably detected.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid composition for detecting respiratory pathogens, characterized in that: Contains at least one of the following primer sets and probe sets: a) A nucleic acid combination 1 for detecting influenza B virus, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 1 to 3; b) a nucleic acid combination 2 for detecting influenza A virus H1N1, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 4 to 6; c) a nucleic acid combination 3 for detecting influenza A virus H3N2, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 7 to 9; d) a nucleic acid combination 4 for detecting the novel coronavirus N gene, comprising a primer probe set having a nucleotide sequence as shown in SEQ ID NOs. 10 to 12; e) Nucleic acid combination 5 for detecting the novel coronavirus O gene, the primer probe set having the nucleotide sequence as shown in SEQ ID NO. 13 to 15; f) a nucleic acid combination 6 for detecting respiratory syncytial virus, a primer probe set having a nucleotide sequence as shown in SEQ ID NOs. 16 to 19; g) a nucleic acid combination 7 for detecting type I parainfluenza virus, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 20 to 22; h) a nucleic acid combination 8 for detecting type II parainfluenza virus, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 23 to 25; i) a nucleic acid combination 9 for detecting type III parainfluenza virus, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 26 to 28; j) a nucleic acid assembly 10 for detecting type IV parainfluenza virus, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 29 to 31; k) a nucleic acid assembly 11 for detecting rhinovirus, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 32 to 36; 1) A nucleic acid combination 12 for detecting adenovirus, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 37 to 39; m) a nucleic acid combination 13 for detecting Haemophilus influenzae, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 40 to 42; n) a nucleic acid combination 14 for detecting Streptococcus pneumoniae, comprising a primer probe set having nucleotide sequences as shown in SEQ ID NOs. 43 to 45; o) A nucleic acid combination 15 for detecting Staphylococcus aureus, comprising a primer probe set with nucleotide sequences such as SEQ ID NOs. 46 to 48.

2. The nucleic acid composition according to claim 1, characterized in that Including at least one of the following (A) to (E): (A) the nucleic acid combination 1 for detecting influenza B virus and the nucleic acid combination 12 for detecting adenovirus; (B) the nucleic acid combination 2 for detecting influenza A virus H1N1 and the nucleic acid combination 14 for detecting Streptococcus pneumoniae; (C) the nucleic acid combination 6 for detecting respiratory syncytial virus and the nucleic acid combination 13 for detecting Haemophilus influenzae; (D) the nucleic acid combination 4 for detecting the novel coronavirus N gene, the nucleic acid combination 7 for detecting type I parainfluenza virus, the nucleic acid combination 8 for detecting respiratory type II parainfluenza virus, and the nucleic acid combination 10 for detecting respiratory type IV parainfluenza virus; (E) The nucleic acid combination 5 for detecting the novel coronavirus O gene and the nucleic acid combination 15 for detecting Staphylococcus aureus.

3. The nucleic acid composition according to claim 1, characterized in that Also included is a combination of nucleic acids for detecting an internal reference; Optionally, the internal reference nucleic acid combination for detecting is an internal reference nucleic acid combination 16 for detecting a β-Actin internal reference gene, including a primer probe set having a nucleotide sequence as shown in SEQ ID NOs. 49 to 51.

4. The nucleic acid composition according to any one of claims 1 to 3, characterized in that The probe is labeled with a luminescent group and a quencher group, the luminescent group is labeled at the 5' end of the probe, and / or the quencher group is labeled at the 3' end of the probe; Optionally, the luminescent group includes quantum dots, FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, TexasRed or Cy5; Optionally, the quencher group includes BHQ1, BHQ2, BHQ3 or MGB.

5. The nucleic acid composition according to any one of claims 1 to 3, characterized in that The working concentration of each forward primer is independently 0.1 to 0.6 μM; the working concentration of the reverse primer is 0.1 to 0.6 μM; Optionally, the working concentration of each forward primer is independently 0.4 μM, and the working concentration of each reverse primer is 0.4 μM.

6. The nucleic acid composition according to claim 5, characterized in that The working concentration of each probe was 0.05–0.4 μM; Optionally, the working concentration of each probe is 0.2 μM.

7. Use of the nucleic acid composition according to any one of claims 1 to 6 in the preparation of a product for the detection of respiratory pathogens.

8. A kit for detecting respiratory pathogens, characterized in that: A nucleic acid composition comprising any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that Also includes one or more of an enzyme mixture, a PCR reaction buffer, dNTPs, a negative control, a positive control, and a blank control; Optionally, the enzyme mixture includes reverse transcriptase, Taq enzyme, and UNG enzyme; Optionally, the buffer comprises a buffer, magnesium ions and a protective agent.

10. Use of the nucleic acid composition according to any one of claims 1 to 6, or the kit according to any one of claims 8 to 9, in detecting respiratory pathogens for non-diagnostic and therapeutic purposes.

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