Respiratory pathogen PCR-HRM detection kit and application thereof

Abstract

The invention discloses a respiratory tract pathogen PCR-HRM detection kit and application thereof, and belongs to the technical field of detection, and the kit comprises primer probes as shown in SEQ ID No.1-90. Based on a PCR-HRM (Polymerase Chain Reaction-High Resolution Melting) method, the detection kit for detecting the 26 common pathogens of the respiratory tract syndrome, established by the invention, has the detection sensitivity of 100 Copies / mL (preferably 50 Copies / mL), and can meet the expected requirement of monitoring the pathogens at a port. In the correctness verification process, compared with a tNGS detection result, the conformity of the detected target species is not lower than 96.1%, which indicates that the method has high reliability and accuracy in pathogen detection.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a PCR-HRM detection kit for respiratory pathogens and its application. Background Technology

[0002] Respiratory infections pose a significant challenge to global public health. According to the World Health Organization (WHO), lower respiratory tract infections are among the deadliest infectious diseases worldwide. These infections can be caused by various pathogens, including bacteria, viruses, and fungi, with bacterial lower respiratory tract infections being particularly noteworthy. Eight bacteria—Legionella pneumophila (LP), Escherichia coli (E. coli), Enterobacter cloacae (EC), Serratia marcescens (SMar), Klebsiella pneumoniae (KO) / Klebsiella pneumoniae (KA), Burkholderia cepacia (BC), and Stenotrophomonas maltophilia (SMA)—are commonly found in lower respiratory tract infections and pose a significant threat to patient health. Therefore, a rapid, efficient, and low-cost detection method for multiple respiratory pathogens is crucial for clinical applications.

[0003] Currently, detection methods for various respiratory pathogens have been developed to some extent. For example, CN107937578A discloses a primer-probe combination for the combined detection of 15 respiratory pathogens, including Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Chlamydia pneumoniae, Mycobacterium tuberculosis, Stenotrophomonas maltophilia, Acinetobacter baumannii, Mycoplasma pneumoniae, Enterococcus faecalis, Legionella pneumophila, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycobacterium abscessus. Although this method can detect 15 pathogens, its limitation is that a single system can only detect 4 respiratory pathogens, and it cannot detect combinations of more than 4 pathogens in one tube. In addition, CN120272616A discloses a probe set for detecting respiratory tract infection pathogens, wherein the pathogens include Haemophilus influenzae, Stenotrophomonas maltophilia, Enterobacter cloacae, Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus parainfluenzae, Moraxella catarrhalis, Burkholderia cepacia, Klebsiella acidogenic, Klebsiella pneumoniae, and others. The detection targets include: *Citrobacter lauridis*, *Serratia marcescens*, *Proteus mirabilis*, Mycobacterium tuberculosis complex, *Mycobacterium avium*, *Mycobacterium intracellulare*, *Legionella pneumophila*, and *Listeria monocytogenes*; *Aspergillus fumigatus*, *Candida albicans*, *Cryptococcus neoformans*, and *Candida auris*; influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, adenovirus, rhinovirus, various coronaviruses, human metapneumovirus, bocavirus, *Mycoplasma pneumoniae*, *Chlamydia pneumoniae*, and *Chlamydia psittaci*. Although this method can detect a variety of common pathogens, it does not employ multiplex fluorescent PCR but rather complex high-throughput sequencing.

[0004] For example, Chinese patent application CN121406832A discloses a nucleic acid combination product for detecting multiple respiratory pathogens, a kit containing the product, and a method. Based on TaqMan's quantitative PCR technology, this nucleic acid combination product enables the detection of seven targets in a single tube, significantly reducing operator time and effort, and offering convenience, speed, and reduced error. The results are also easier to interpret. However, the number of targets detected by this patent application is still limited, and its detection sensitivity needs further improvement. Summary of the Invention

[0005] This invention aims to develop a PCR-HRM detection kit and its applications for 26 common pathogens causing respiratory syndromes. This invention utilizes PCR amplification combined with molecular beacon melting curve technology to determine the pathogen type based on the DNA melting temperature (Tm), resulting in accurate results and enabling high-throughput detection with multiple assays per tube (10-30 assays per tube). The PCR-HRM method targets the specific conserved sequences of each pathogen as the target region, designing specific primers and probes to accurately identify the target pathogen. The target detection limit is no higher than 100 copies / mL, preferably no more than 50 copies / mL, covering low concentrations of pathogen nucleic acids.

[0006] One aspect of the present invention relates to a PCR-HRM detection kit for respiratory pathogens, the kit comprising the primers and probes shown in SEQ ID No. 1-90.

[0007] In a preferred embodiment of the present invention, the kit further includes 2M folic acid. By introducing folic acid into the kit, the present invention can further improve the detection sensitivity of the kit.

[0008] In a preferred embodiment of the present invention, the kit further includes PCR buffer, MgCl2, dNTPs, Hot-start Taq polymerase, and reverse transcriptase.

[0009] In a preferred embodiment of the present invention, the 5' end of the probe is labeled with a fluorescent group; preferably, the fluorescent group is selected from FAM, HEX, ROX and CY5.

[0010] Another aspect of the present invention relates to the application of the above-described kit in the detection of respiratory pathogens.

[0011] In a preferred embodiment of the present invention, the respiratory pathogens include rubella virus, Corynebacterium diphtheriae, Bordetella pertussis, Glandella glanders, monkeypox virus, Group A Streptococcus, Bordetella parapertussis, mumps virus, Legionella, Neisseria meningitidis, coronavirus, enterovirus, norovirus, measles virus, Haemophilus influenzae, Mycobacterium tuberculosis complex, novel coronavirus, influenza A virus, human metapneumovirus, Middle East respiratory syndrome coronavirus, varicella-zoster virus, respiratory syncytial virus, rhinovirus, Streptococcus pneumoniae, smallpox virus, and influenza B virus.

[0012] Based on the PCR-HRM method, this invention establishes a detection kit for 26 common pathogens causing respiratory syndromes, with a detection sensitivity of 100 copies / mL (preferably 50 copies / mL), which meets the expected requirements for pathogen monitoring at ports of entry. During accuracy verification, compared with tNGS detection results, the concordance rate of the detected target species was no less than 96.1%, indicating that this invention has high reliability and accuracy in pathogen detection. Detailed Implementation

[0013] The following description is based on preferred embodiments of the present invention, but the present invention is not limited thereto.

[0014] Example 1:

[0015] 1.1 Instruments

[0016] Real-time PCR instruments: QuantGene 9600 Real-time PCR instrument, ABI Q5 Real-time PCR instrument. Automated nucleic acid extractor: Xi'an Tianlong GeneRotex96 Automated Nucleic Acid Extractor.

[0017] 1.2 Primer Design

[0018] Primer and probe design primarily targets specific gene regions of each detection target. Whole genome sequences and housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) sequences for each species were obtained from RefSeq and GenBank databases. Homology verification of any 30-mer short fragment within the designed region was then performed using BlastN software. Primers were designed using Primer3 software based on the homology-screened short fragments, and output after inclusion verification across genomes of the same HPV type. The designed primers and probes exhibited sufficient specificity among different HPV types and were relatively conserved across many different strains of the same type. Primers and probes were designed and produced for each type. The sequences listed in Table 1 were selected after verification of amplification effect and sensitivity. Primers and probes were synthesized by Hunan Aikerui Biotechnology Co., Ltd. and Shanghai Sangon Biotech Co., Ltd. Internal control primers and probes specifically amplified a 100bp fragment of the housekeeping gene GAPDH in human cervical cells for sample quality testing. Quality control was performed after synthesis to ensure that the purity and concentration met experimental requirements.

[0019] Table 1 Primer and probe sequences for each target

[0020]

[0021]

[0022] 1.3 Virus strains and clinical samples

[0023] The remaining 155 samples submitted to a testing institution for tNGS testing between June and September 2024 included 90 nasopharyngeal swab samples and 65 bronchoalveolar lavage fluid samples. All samples were consistent with the tNGS test results, as detailed in Table 2.

[0024] 1.4 Nucleic acid extraction

[0025] For nucleic acid extraction from the samples, automated nucleic acid extraction reagents and equipment were employed to ensure the accuracy, efficiency, and stability of the experimental results. The Xi'an Tianlong GeneRotex96 automated nucleic acid extractor and its matching nucleic acid extraction or purification reagents were used. This equipment achieves automated nucleic acid extraction based on the magnetic bead method, offering advantages such as ease of operation, high throughput, and high extraction purity. The matching reagents were optimized to efficiently lyse cells and specifically bind to nucleic acids, effectively removing impurities and inhibitors. Specific operations were performed according to the nucleic acid extraction reagent instructions to ensure standardized extraction processes and avoid nucleic acid degradation and cross-contamination. Extracted nucleic acid samples can be immediately used for subsequent experiments or stored at -20℃ for later use.

[0026] The diluted reference sample was subjected to RNA / DNA extraction following the procedures described above.

[0027] 1.5 Detection System and Reaction Conditions

[0028] Detection system A: 10µL of 1×PCR buffer (Takara Bio Engineering (Dalian) Co., Ltd.), 2mM MgCl2 (Promega, USA), 5µL of 0.2mM dNTPs (Takara Bio Engineering (Dalian) Co., Ltd.), 2µL of 37.5nM primers F for each target, 2µL of 500nM primers R for each target, 2µL of 25nM probes MB for each target, 1µL of Hot-start Taq polymerase (Takara Bio Engineering (Dalian) Co., Ltd.), 1µL of reverse transcriptase (Takara Bio Engineering (Dalian) Co., Ltd.), and 1µL of extracted RNA / DNA as template, with ddH2O added to make up to 40µL of reaction system.

[0029] Detection system B: Same as detection system A, except that 2µL of 2mM folic acid is added.

[0030] 1.6 Sensitivity / Detection Limit Test

[0031] A recombinant plasmid of a certain pathogen was serially diluted 10-fold to select a concentration of 10. 3 10 2 Sensitivity tests were conducted on recombinant plasmids with concentrations of 1, 50, and 20 copies / mL, with a negative control set up to evaluate the sensitivity of the established method. The results are shown in Tables 2 and 3. The experimental results show that the detection limit of system A of the present invention is 100 copies / mL, while the detection limit of system B is 50 copies / mL.

[0032] Table 2 Validation results of the lowest detection limit of system A

[0033]

[0034] Table 3 Validation results of the lowest detection limit for System B

[0035]

[0036] 1.7 Specificity Test

[0037] Other pathogen references not within the detection range were selected (as shown in Table 4), and the established detection system was used for detection and analysis. A negative control was also set up to evaluate the specificity of the established method.

[0038] Table 4. Analytical specificity verification sample information and results for System B.

[0039]

[0040] 1.8 Accuracy Test

[0041] The accuracy of this method was verified using a clinical comparative trial. Initially, the established method was used to test 155 collected clinical samples. The positive detection rate and the detection status of each microorganism were statistically analyzed and compared with the tNGS results. Kappa consistency analysis was also performed (Kappa > 0.8 indicates high consistency). Specific detection and comparison results are shown in Table 5.

[0042] Table 5 Comparison of PCR-melting curve method (System B) and tNGS detection results

[0043]

[0044] Based on the PCR-HRM method, this invention establishes a detection kit for 26 common pathogens causing respiratory syndromes, with a detection sensitivity of 100 copies / mL (preferably 50 copies / mL), which meets the expected requirements for pathogen monitoring at ports of entry. During accuracy verification, compared with tNGS detection results, the concordance rate of the detected target species was no less than 96.1%, indicating that this invention has high reliability and accuracy in pathogen detection.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A PCR-HRM detection kit for respiratory pathogens, characterized in that... The kit includes the primers and probes shown in SEQ ID No. 1-90.

2. The reagent kit according to claim 1, characterized in that... The kit also includes 2M folic acid.

3. The kit according to claim 1 or 2, characterized in that... The kit also includes PCR buffer, MgCl2, dNTPs, Hot-start Taq polymerase, and reverse transcriptase.

4. The kit according to claim 1 or 2, characterized in that... The probe is labeled with a fluorescent group at its 5' end.

5. The reagent kit according to claim 4, characterized in that... The fluorescent group is selected from FAM, HEX, ROX and CY5.

6. The use of the kit according to any one of claims 1-5 in the detection of respiratory pathogens.

7. The application according to claim 6, characterized in that... The respiratory pathogens mentioned include rubella virus, Corynebacterium diphtheriae, Bordetella pertussis, Glandella glanders, monkeypox virus, Group A Streptococcus, Bordetella parapertussis, mumps virus, Legionella, Neisseria meningitidis, coronavirus, enterovirus, norovirus, measles virus, Haemophilus influenzae, Mycobacterium tuberculosis complex, novel coronavirus, influenza A virus, human metapneumovirus, Middle East respiratory syndrome coronavirus, varicella-zoster virus, respiratory syncytial virus, rhinovirus, Streptococcus pneumoniae, smallpox virus, and influenza B virus.

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