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CN-122012814-A - Respiratory pathogen PCR-HRM detection kit and application thereof

CN122012814ACN 122012814 ACN122012814 ACN 122012814ACN-122012814-A

Abstract

The invention discloses a PCR-HRM detection kit for respiratory pathogens and application thereof, belonging to the technical field of detection, wherein the kit comprises primer probes shown in SEQ ID No. 1-90. Based on a PCR-HRM method, the detection kit for detecting 26 common pathogens of respiratory tract syndrome, which is established by the invention, has the detection sensitivity of 100 Copies/mL (preferably 50 Copies/mL) and can meet the expected requirements of port monitoring pathogens. In the process of verifying the correctness, compared with tNGS detection results, the detected target species coincidence degree is not lower than 96.1%, which shows that the method has higher reliability and accuracy in pathogen detection.

Inventors

  • HAN HUANMEI
  • LIU BAOHUA
  • BAI LI
  • WANG ZHILING
  • ZHANG YAQI
  • BI JING

Assignees

  • 济南国际旅行卫生保健中心(济南海关口岸门诊部)

Dates

Publication Date
20260512
Application Date
20260213

Claims (7)

  1. 1. A respiratory pathogen PCR-HRM detection kit is characterized by comprising primer probes shown in SEQ ID No. 1-90.
  2. 2. The kit according to claim 1, wherein the kit further comprises 2M folic acid.
  3. 3. The kit according to claim 1 or 2, wherein the kit further comprises PCR buffer, mgCl 2 , dNTPs, hot-start Taq polymerase and reverse transcriptase.
  4. 4. The kit according to claim 1 or 2, wherein the 5' -end of the probe is labeled with a fluorescent group.
  5. 5. The kit according to claim 4, wherein the fluorescent group is selected from FAM, HEX, ROX and CY5.
  6. 6. Use of a kit according to any one of claims 1-5 for detecting respiratory pathogens.
  7. 7. The method of claim 6, wherein the respiratory pathogen comprises rubella virus, corynebacterium diphtheriae, bordetella pertussis, bacillus meldonium, simian poxvirus, streptococcus group A, paraquaticus pertussis, mumps virus, legionella, neisseria meningitidis, coronavirus, enterovirus, north Rubi virus, haemophilus influenzae, mycobacterium tuberculosis complex, novel coronavirus, influenza A virus, human metapneumovirus, central east respiratory syndrome coronavirus, varicella-zoster virus, respiratory syncytial virus, rhinovirus, streptococcus pneumoniae, smallpox virus, and influenza B virus.

Description

Respiratory pathogen PCR-HRM detection kit and application thereof Technical Field The invention belongs to the technical field of detection, and particularly relates to a PCR-HRM detection kit for respiratory pathogens and application thereof. Background Respiratory tract infections are a major challenge in the world public health sector, and lower respiratory tract infections are reported by the World Health Organization (WHO) to be the leading cause of world-deadly infectious diseases. Such infections may be caused by a variety of pathogens, bacteria, viruses, fungi, etc., with bacterial lower respiratory tract infections being of particular concern. The 8 bacteria of Legionella Pneumophila (LP), escherichia coli (E.coli), enterobacter Cloacae (EC), serratia marcescens (SMar), klebsiella Oxytoca (KO)/Klebsiella oxytoca (KA), burkholderia Cepacia (BC) and Zygomonas maltophilia (SMA) are more in lower respiratory tract infection and have a great health hazard to patients. Therefore, the detection method capable of rapidly, efficiently and low-cost detecting various respiratory pathogens is particularly important in clinical application. At present, a plurality of detection methods for respiratory tract pathogenic bacteria have been developed. For example, CN107937578a discloses primer probe combinations for joint detection of 15 respiratory tract infection 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 abscess. Although the method can detect 15 pathogens, the limitation is that a single system can only detect 4 respiratory pathogens, and cannot detect a combination mode of more than 4 pathogens in a 1-tube way. In addition, CN120272616a discloses a probe set for detecting pathogenic microorganisms of respiratory tract infection, wherein the pathogenic microorganisms include haemophilus influenzae, stenotrophomonas maltophilia, enterobacter cloacae, streptococcus pyogenes, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, streptococcus pneumoniae, haemophilus parainfluenza, moraxella catarrhalis, burkholderia cepacia, klebsiella oxytoca, klebsiella aerogenes, citric acid bacillus fradiae, serratia marcescens, proteus mirabilis, mycobacterium tuberculosis complex, mycobacterium avium, mycobacterium intracellulare, legionella pneumophila, listeria monocytogenes, aspergillus fumigatus, candida albicans, novel cryptococcus, candida auricularis, influenza a virus, influenza b virus, respiratory syncytial virus, parainfluenza virus, adenovirus, rhinopneumovirus, various coronaviruses, human metapneumovirus, bocavirus, mycoplasma, and chlamydia psilosis. Although this method is capable of detecting a wide variety of common pathogens, it does not employ multiplex fluorescent PCR, but rather complex high throughput sequencing. For example, chinese patent application CN121406832a discloses a nucleic acid combination product for detecting multiple respiratory pathogens, a kit and a method comprising the same, and the nucleic acid combination product realizes that 7 targets can be detected by one tube based on the technique of Taqman fluorescence quantification, greatly reduces the time and effort of operators, is convenient and quick, is not easy to make mistakes, and is simpler and easier to understand in result interpretation. However, the targets detected by this patent application are still limited, and the detection sensitivity thereof is still to be further improved. Disclosure of Invention The invention aims at developing a kit for detecting 26 common pathogens of respiratory tract syndrome by adopting a PCR-HRM method and application thereof. The invention judges the type of pathogenic microorganism according to the DNA melting temperature (Tm) by combining PCR amplification with a molecular beacon melting curve technology, has accurate result, and can realize high-throughput detection of one tube for multiple detection (10-30 detection can be realized by one tube). The PCR-HRM method detects specific conserved sequences of pathogenic microorganisms as target areas, and designs specific primers and probes, so that target pathogens can be accurately identified, the minimum detection limit is not higher than 100copies/mL, preferably not more than 50copies/mL, and low-concentration pathogen nucleic acid can be covered. One aspect of the invention relates to a respiratory pathogen PCR-HRM detection kit, which comprises primer probes shown in SEQ ID No. 1-90. In a preferred embodiment of the invention, the kit further comprises 2M folic acid. According to the invention, folic acid is introduced into the kit, so that the detection s