Primer and probe combination for joint detection of seven pathogens, kit and application

Abstract

The invention relates to the technical field of biological detection, in particular to a primer and probe combination for joint detection of seven pathogens, a kit and application. The primer and probe combination comprises primers and probes for specifically detecting the following pathogens: novel coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, rhinovirus and adenovirus, and nucleotide sequences are shown as SEQ ID NO: 1-24. Based on the primer and probe combination of the multiple system, seven targets can be simultaneously detected in one sample within 30 minutes. In addition, the sensitivity of the multiple systems is high, and the lowest detection limit is 500 copies / mL; and the kit has good specificity and has no cross reaction with other pathogens causing the same symptom. In addition, a PCR reagent comprising the primer and probe combination can be freeze-dried, a nucleic acid elution product to be detected is adopted to redissolve the PCR reagent during detection, and closed amplification is performed on a micro-fluidic chip.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a primer and probe combination, kit, and application for the joint detection of seven pathogens. Background Technology

[0002] Respiratory infections are among the most common infectious diseases worldwide, causing upper respiratory tract infections (such as the common cold) or lower respiratory tract infections (such as bronchitis and pneumonia). Pneumonia is one of the most serious complications of respiratory infections, posing a significant threat, especially to infants, the elderly, and those with weakened immune systems. Therefore, the etiological diagnosis of respiratory infections is increasingly important in clinical practice.

[0003] Viral etiological diagnosis helps clinicians assess disease progression and prognosis. For certain viral infections, such as adenovirus, respiratory syncytial virus, and influenza virus, specific antiviral drugs can be used. Studies have shown that viral etiological diagnosis plays a crucial role in clinical treatment; for example, early anti-influenza treatment can not only control disease progression but also significantly improve the prognosis of patients with severe influenza. Currently, commonly used clinical methods for detecting respiratory pathogens mainly include culture methods, antigen detection, serological detection, and molecular biological detection.

[0004] However, some commonly used clinical methods have the following shortcomings: culture methods generally take several days to two weeks, which is cumbersome and time-consuming, with low diagnostic efficiency and limited clinical application; antigen detection methods are convenient and quick to use, but have poor sensitivity; serological detection methods, when used to detect respiratory viruses, have disadvantages such as a window period for antibodies and false positives, so they are usually only used as a supplementary diagnostic method.

[0005] Molecular biological detection methods have become an important tool for diagnosing respiratory viral infections. Based on viral gene sequences, assays can be rapidly established to quickly address the need for etiological diagnosis during infectious disease outbreaks. This significantly improves the sensitivity of respiratory viral infection detection, providing results within hours. It solves the problems of difficult and lengthy traditional virus culture methods, and also overcomes the low positive rate of traditional immunological methods. Summary of the Invention

[0006] The purpose of this invention is to provide a primer and probe combination, kit, and application for the joint detection of seven pathogens, which can simultaneously identify multiple respiratory pathogens (including influenza A virus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, rhinovirus, adenovirus, and novel coronavirus) with good specificity and sensitivity.

[0007] Therefore, in a first aspect, the present invention provides a primer and probe combination for the joint detection of seven pathogens, comprising: Primers for detecting the novel coronavirus have nucleotide sequences as shown in SEQ ID NO: 1~2 and SEQ ID NO: 4~5, and probes for detecting the novel coronavirus have nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 6. Primers for detecting influenza A virus have nucleotide sequences as shown in SEQ ID NO: 7-8, and probes for detecting influenza A virus have nucleotide sequences as shown in SEQ ID NO: 9. Primers for detecting influenza B virus have nucleotide sequences as shown in SEQ ID NO: 10-11, and probes for detecting influenza B virus have nucleotide sequences as shown in SEQ ID NO: 12. Primers for detecting respiratory syncytial virus (RSV) have nucleotide sequences as shown in SEQ ID NO: 13-14, and probes for detecting RSV have nucleotide sequences as shown in SEQ ID NO: 15. The primers for detecting Mycoplasma pneumoniae have nucleotide sequences as shown in SEQ ID NO: 16~17, and the probes for detecting Mycoplasma pneumoniae have nucleotide sequences as shown in SEQ ID NO: 18. Primers for detecting rhinovirus have nucleotide sequences as shown in SEQ ID NO: 19-20, and probes for detecting rhinovirus have nucleotide sequences as shown in SEQ ID NO: 21. Primers for detecting adenovirus have nucleotide sequences as shown in SEQ ID NO: 22-23, and probes for detecting adenovirus have nucleotide sequences as shown in SEQ ID NO: 24. The probe is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.

[0008] In some embodiments, the primer and probe combination further includes a primer for detecting an internal standard, the nucleotide sequence of which is shown in SEQ ID NO: 25-26, and a probe for detecting an internal standard, the nucleotide sequence of which is shown in SEQ ID NO: 27.

[0009] In some embodiments, the fluorescent group includes at least one selected from the group consisting of FAM, ROX, Cy5, Cy5.5, CY7, VIC, AF1, and AF2; the quenching group includes BHQ.

[0010] A second aspect of the present invention provides a kit comprising the primer and probe combination described in the first aspect of the present invention, dNTPs, PCR buffer, and DNA polymerase.

[0011] In some embodiments, the kit further includes a microfluidic chip and lyophilized powder; the lyophilized powder includes the primer and probe combination, dNTPs, PCR buffer, and DNA polymerase; The microfluidic chip is adapted to contain the lyophilized powder, which can be reconstituted by the sample solution to be tested and subjected to PCR amplification reaction.

[0012] A third aspect of the present invention provides a PCR reaction system comprising a sample to be tested, the primer and probe combination described in the first aspect of the present invention, dNTPs, PCR buffer, and DNA polymerase.

[0013] In some embodiments, the concentration of the primers in the PCR reaction system is 300nM to 500nM for each primer, for example, about 300nM, 400nM, 500nM, etc.; and the concentration of the probes is 150nM to 250nM for each probe, for example, about 150nM, 200nM, 250nM, etc.

[0014] In some embodiments, the DNA polymerase includes at least one of the following: Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, or Tgo DNA polymerase.

[0015] A fourth aspect of the present invention provides the application of the primer and probe combination described in the first aspect of the present invention, the kit described in the second aspect of the present invention, and the PCR reaction system described in the third aspect of the present invention in the preparation of a method for detecting respiratory infectious pathogens.

[0016] In some embodiments, the respiratory pathogens include at least one of the following: novel coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, rhinovirus, and adenovirus.

[0017] In some embodiments, the detection limit concentration for detecting the respiratory infection pathogen is 500 copies / mL.

[0018] Compared with the prior art, the technical solution of this application has the following beneficial effects: This invention establishes a highly sensitive and specific molecular biological detection method based on real-time quantitative PCR for typical respiratory pathogens such as novel coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, rhinovirus, and adenovirus. The invention provides related primer and probe combinations, kits, and detection methods. Using the primer and probe combinations provided by this invention for pathogen detection simplifies the cumbersome gel electrophoresis verification steps required for conventional PCR, and offers advantages such as high sensitivity, strong specificity, and the ability to simultaneously detect multiple targets. Detailed Implementation

[0019] Exemplary embodiments of this disclosure will now be described in more detail. It should be understood that this disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of this disclosure to those skilled in the art.

[0020] This invention employs the TaqMan probe method, designing specific primers and probes for specific sequences of influenza A virus, enabling universal detection of various subtypes of influenza A virus, not limited to H1N1, H3N2, H5N1, and H7N9; specific primers and probes for specific sequences of influenza B virus, enabling universal detection of various subtypes of influenza B virus, not limited to Victoria and Yamagata lineages; specific primers and probes for specific sequences of Mycoplasma pneumoniae, enabling universal detection of various subtypes of Mycoplasma pneumoniae, not limited to type I and type II; and specific primers and probes for specific sequences of respiratory syncytial virus. The probes can be used for the universal detection of various subtypes of respiratory syncytial virus (RSV), not limited to subtypes A and B; specific primers and probes have been designed for rhinovirus specific sequences, which can be used for the universal detection of various rhinovirus subgroups, not limited to subtypes A, B, and C; specific primers and probes have been designed for adenovirus specific sequences, which can be used for the universal detection of various adenovirus subgroups, not limited to subtypes 1, 2, 3, 4, 5, 7, and 55; specific primers and probes have been designed for novel coronavirus specific sequences, which can be used for the universal detection of various variant strains of the novel coronavirus, not limited to alpha variants, beta variants, delta variants, and omega-Jon variants.

[0021] This invention also selects the human RP gene as an internal reference detection target. During the sampling process, a template is obtained from the sample, which participates in the entire process of nucleic acid extraction and PCR amplification, thereby enabling monitoring of the entire process.

[0022] To achieve the above objectives, this invention designs specific primers (denoted by F for upstream primer and R for downstream primer) and probes (denoted by P), as shown in Table 1: Table 1 Primer and probe sequences

[0023] Each probe has a fluorescent group labeled at its 5' end and a quenching group labeled at its 3' end.

[0024] In some embodiments, the fluorescent group is selected from FAM, ROX, Cy5, Cy5.5, CY7, VIC, AF1, AF2, etc.; the quenching group is BHQ.

[0025] In some embodiments, a kit is provided that includes dNTPs, PCR buffer, DNA polymerase, and primer and probe combinations provided in embodiments of the present invention.

[0026] The following description provides examples of the invention, thereby making the advantages and various effects of the invention more clearly apparent. Those skilled in the art will understand that these examples are illustrative and not intended to limit the invention. Experimental methods described in the following detailed embodiments, unless otherwise specified, generally follow conventional methods and conditions of molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or the conditions recommended by the manufacturer.

[0027] Example 1 This embodiment uses the primer and probe combinations shown in Table 1. Specifically, the 5' end of the probes OP and NP for detecting the novel coronavirus is labeled with the fluorescent group FAM, and the 3' end with the quencher group BHQ; the 5' end of the probe AP for detecting influenza A virus is labeled with the fluorescent group VIC, and the 3' end with the quencher group BHQ; the 5' end of the probe BP for detecting influenza B virus is labeled with the fluorescent group CY5, and the 3' end with the quencher group BHQ; and the 5' end of the probe RSV-P for detecting respiratory syncytial virus is labeled with the fluorescent group CY5. 5.5 The probe MP-P for detecting Mycoplasma pneumoniae is labeled with the fluorescent group ROX at the 5' end and the quencher group BHQ at the 3' end; the probe RV-P for detecting rhinovirus is labeled with the fluorescent group CY7 at the 5' end and the quencher group BHQ at the 3' end; the probe ADV-P for detecting adenovirus is labeled with the fluorescent group AF1 at the 5' end and the quencher group BHQ at the 3' end; the probe IC-P for detecting internal standards is labeled with the fluorescent group AF2 at the 5' end and the quencher group BHQ at the 3' end.

[0028] The PCR reaction solution was prepared using the primer and probe combinations described above, as shown in Table 2. Table 2 PCR reaction solution

[0029] The PCR reaction procedure is as follows: 95℃, 2 minutes, 1 cycle; 95℃, 5 seconds, 60℃, 30 seconds, 40 cycles, collect fluorescence.

[0030] Three samples were used: influenza A virus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, rhinovirus, adenovirus, and novel coronavirus. These samples were diluted to 3000, 2000, 1000, 500, 250, 100, and 50 copies / mL, respectively. Using the above-mentioned PCR reaction solution, each concentration was tested 20 times. The Ct value was calculated, and the detection rate was determined. The concentration corresponding to a detection rate ≥95% was the limit of detection (LOD). The detection results are shown in Tables 3-8.

[0031] Table 3. Results of Influenza A Virus Detection

[0032] Table 4. Influenza B virus detection results

[0033] Table 5. Respiratory syncytial virus (RSV) detection results

[0034] Table 6. Mycoplasma pneumoniae detection results

[0035] Table 7 Rhinovirus Detection Results

[0036] Table 8 Adenovirus Detection Results

[0037] Table 9. Novel Coronavirus Detection Results

[0038] The above results indicate that the detection limit concentration of this primer-probe combination is 500 copies / mL.

[0039] Example 2 This embodiment, based on Embodiment 1, further conducts cross-reactivity testing. Pathogens with similar clinical symptoms and the same infection location as the target were detected. Specific pathogens tested are shown in Table 9, where the concentration of pathogen samples numbered 1-15 is 10.5 PFU / mL, the concentration of pathogen samples numbered 16-35 was 10. 6 PFU / mL.

[0040] Table 9 Cross-reactivity test results

[0041] The above results demonstrate that the primer and probe combination provided by this invention exhibits no cross-reactivity with the aforementioned pathogens when used for pathogen detection. The primer and probe combination provided by this invention possesses good specificity.

[0042] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A primer and probe combination for simultaneous detection of seven pathogens, characterized in that, Comprise: primers for detecting the novel coronavirus, the nucleotide sequence of which is shown as SEQ ID NO: 1~2, SEQ ID NO: 4~5, probes for detecting the novel coronavirus, the nucleotide sequence of which is shown as SEQ ID NO: 3, SEQ ID NO: 6; primers for detecting influenza A virus, the nucleotide sequence of which is shown as SEQ ID NO: 7~8, probes for detecting influenza A virus, the nucleotide sequence of which is shown as SEQ ID NO: 9; primers for detecting influenza B virus, the nucleotide sequence of which is shown as SEQ ID NO: 10~11, probes for detecting influenza B virus, the nucleotide sequence of which is shown as SEQ ID NO: 12; primers for detecting respiratory syncytial virus, the nucleotide sequence of which is shown as SEQ ID NO: 13~14, probes for detecting respiratory syncytial virus, the nucleotide sequence of which is shown as SEQ ID NO: 15; primers for detecting mycoplasma pneumoniae, the nucleotide sequence of which is shown as SEQ ID NO: 16~17, probes for detecting mycoplasma pneumoniae, the nucleotide sequence of which is shown as SEQ ID NO: 18; primers for detecting rhinovirus, the nucleotide sequence of which is shown as SEQ ID NO: 19~20, probes for detecting rhinovirus, the nucleotide sequence of which is shown as SEQ ID NO: 21; primers for detecting adenovirus, the nucleotide sequence of which is shown as SEQ ID NO: 22~23, probes for detecting adenovirus, the nucleotide sequence of which is shown as SEQ ID NO: 24; The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

2. The primer and probe combination of claim 1, wherein, It also comprises primers for detecting internal standards, the nucleotide sequence of which is shown as SEQ ID NO: 25~26, probes for detecting internal standards, the nucleotide sequence of which is shown as SEQ ID NO:

27.

3. The primer and probe combination of claim 1 or 2, wherein The fluorescent group comprises at least one selected from the group consisting of FAM, ROX, Cy5, Cy5.5, CY7, VIC, AF1, AF2; the quenching group comprises BHQ.

4. A kit characterized in that, It comprises the primer and probe combination of any one of claims 1~3, dNTPs, PCR buffer and DNA polymerase.

5. The kit of claim 4, wherein It also comprises a microfluidic chip and a lyophilized powder; the lyophilized powder comprises the primer and probe combination, dNTPs, PCR buffer and DNA polymerase; The microfluidic chip is suitable for containing the lyophilized powder, which can be reconstituted by the sample solution to be tested, and PCR amplification reaction is carried out.

6. A PCR reaction system, characterized by, It comprises a sample to be tested, the primer and probe combination of any one of claims 1~3, dNTPs, PCR buffer and DNA polymerase.

7. The PCR reaction system according to claim 6, wherein In the PCR reaction system, the concentration of the primer is 300nM~500nM for each primer; the concentration of the probe is 150nM~250nM for each probe.

8. The PCR reaction system according to claim 6, wherein The DNA polymerase comprises at least one of the following group: Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase or Tgo DNA polymerase.

9. Use of the primer and probe combination of any one of claims 1-3, the kit of claim 4 or 5, and the PCR reaction system of any one of claims 6-8 for preparing a detection of a respiratory infection pathogen.

10. Use according to claim 9, wherein the compound is ###0002### The respiratory infection pathogen comprises at least one of the following group: novel coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, Mycoplasma pneumoniae, rhinovirus and adenovirus. Preferably, the detection limit concentration of the respiratory infection pathogen is 500 copies / mL.

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