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CN-121992137-A - Primer probe combination, kit and application for detecting candida otophylla by digital PCR

CN121992137ACN 121992137 ACN121992137 ACN 121992137ACN-121992137-A

Abstract

The invention relates to the field of pathogen detection, in particular to a primer probe combination, a kit and application for detecting candida otophylla by digital PCR. The primer probe combination provided by the invention is a primer probe combination designed by taking the candida auriculata genetic conservation site ITS as an amplified target gene, can be used for detecting candida auriculata through digital PCR, can directly quantitatively detect candida auriculata, can detect the candida auriculata content in a sample to be detected under extremely low copy number, can accurately quantify candida auriculata load in an environmental sample, can be suitable for clinical early diagnosis, hospital feel monitoring and basic quick detection, and provides accurate technical support for prevention and control of multi-drug-resistant candida auriculata.

Inventors

  • REN LEI
  • HAN XIAOQIANG
  • MA XIAOYU
  • GUO TAO

Assignees

  • 北京威泰科生物技术有限公司

Dates

Publication Date
20260508
Application Date
20260224

Claims (10)

  1. 1. A primer probe combination for detecting candida otophylla (Candida auris) is characterized by comprising a primer and a probe for detecting candida otophylla and a primer and a probe for detecting an internal reference gene: The nucleotide sequence of the upstream primer for detecting candida auriculata is shown as SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5; The nucleotide sequence of the upstream primer for detecting the reference gene is shown as SEQ ID NO.7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.8, and the nucleotide sequence of the probe for detecting the reference gene is shown as SEQ ID NO. 9; the probes are marked with fluorescent groups and quenching groups, and the fluorescent groups marked on the probes for detecting candida otophylla and reference genes are different.
  2. 2. The primer probe combination of claim 1, wherein the fluorescent groups labeled on different probes are selected from any one of ATTO, FAM, VIC, ROX, CY or CY 5.5.
  3. 3. The primer probe combination of claim 1, wherein the quenching groups labeled on different probes are selected from any of BHQ1, BHQ2, BHQ3, or MGB.
  4. 4. The primer probe combination according to any one of claims 1to 3, wherein the probe of candida otophylla is labeled with a fluorescent group FAM at the 5 'end and a quenching group BHQ1 at the 3' end, and the probe of the reference gene is labeled with a fluorescent group ROX at the 5 'end and a quenching group BHQ2 at the 3' end.
  5. 5. A kit for detecting candida otophylla, comprising the primer probe combination of any one of claims 1-4.
  6. 6. The kit of claim 5, further comprising a digital PCR reaction buffer and nuclease-free water, wherein the digital PCR reaction buffer comprises one or more of TaqDNA polymerase, UDG enzyme, dNTPs, mg 2+ , and water.
  7. 7. Use of the primer probe combination of any one of claims 1-4 or the kit of claim 5 or 6 for the detection of candida otophylla for non-diagnostic and non-therapeutic purposes.
  8. 8. The use according to claim 7, wherein the method of detecting comprises: Using DNA of a sample to be detected as a template, performing digital PCR amplification by using the primer probe combination according to any one of claims 1-4, collecting fluorescent signals of PCR amplification products, and performing result quantification and judgment analysis; When the fluorescent signal on the probe of the candida auriculata has a positive point, and the fluorescent signal on the probe of the internal reference gene has a positive point, the sample to be tested contains the candida auriculata; And when the fluorescent signal on the probe of the reference gene has no positive point, judging that the detection is abnormal, and retesting is needed.
  9. 9. The method according to claim 8, wherein the working concentration of the different probes is 150-300nM, the working concentration of the upstream primer of the different primers is 300-600nM, and the working concentration of the downstream primer of the different primers is 300-600nM.
  10. 10. The method of claim 8, wherein the reaction sequence for digital PCR amplification comprises 95℃for 5min, 95℃for 20 s,54℃for 30 s, and 45 cycles.

Description

Primer probe combination, kit and application for detecting candida otophylla by digital PCR Technical Field The invention relates to the field of pathogen detection, in particular to a primer probe combination, a kit and application for detecting candida otophylla by digital PCR (dPCR). Background Candida otophylla (Candida auris) is a multi-drug resistant fungal pathogen. However, most medical institutions have not established an active screening mechanism for this pathogen, and actual infection cases may be severely underestimated. Candida otophylla has the capability of surviving on the skin of a patient and the surfaces of objects in the medical environment for a long time, can be transmitted indirectly through direct contact or through the surfaces of polluted environments, and is easy to cause outbreak in a hospital. Currently, common methods of identification of invasive candida include microscopy, histopathological examination, culture methods, and the like. The method has the advantages of low positive rate of microscopic examination and histopathological detection results, possibility of false negative interference, long culture method period, high requirements on technical conditions and professional level of operators, high homology of candida otophylla and candida shikimchi, possible false identification when only using the culture method, and incapacity of distinguishing experimental strains when only judging whether a patient has fungal infection or not by serological detection. It is therefore desirable to develop a method that enables accurate and rapid detection of candida otophylla. Disclosure of Invention In order to solve the problems, the invention provides a primer probe combination, a kit and application for detecting candida otophylla by digital PCR. The primer probe combination provided by the invention can accurately and rapidly detect candida otophylla. In order to achieve the above object, the present invention provides the following technical solutions: the invention provides a primer probe combination for detecting candida otophylla, which comprises a primer and a probe for detecting candida otophylla and a primer and a probe for detecting an internal reference gene: The nucleotide sequence of the upstream primer for detecting candida auriculata is shown as SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5; The nucleotide sequence of the upstream primer for detecting the reference gene is shown as SEQ ID NO.7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.8, and the nucleotide sequence of the probe for detecting the reference gene is shown as SEQ ID NO. 9; the probes are marked with fluorescent groups and quenching groups, and the fluorescent groups marked on the probes for detecting candida otophylla and reference genes are different. Preferably, the fluorescent groups labeled on the different probes are selected from any one of ATTO, FAM, VIC, ROX, CY or CY 5.5. Preferably, the quenching groups labeled on the different probes are selected from any of BHQ1, BHQ2, BHQ3 or MGB. Preferably, the 5 'end of the candida otophylla probe is marked with a fluorescent group FAM, the 3' end of the candida otophylla probe is marked with a quenching group BHQ1, the 5 'end of the reference gene probe is marked with a fluorescent group ROX, and the 3' end of the reference gene probe is marked with a quenching group BHQ2. The invention provides a kit for detecting candida otophylla, which comprises the primer probe combination in the technical scheme. Preferably, the kit further comprises a digital PCR reaction buffer solution and water without a nuclease, wherein the digital PCR reaction buffer solution comprises one or more of Taq DNA polymerase, UDG enzyme, dNTPs, mg 2+ and water. The invention provides application of the primer probe combination or the kit in detection of candida otophylla for non-diagnosis and non-treatment purposes. Preferably, the method of detecting comprises: Taking DNA of a sample to be detected as a template, carrying out digital PCR amplification by utilizing the primer probe combination in the technical scheme, collecting fluorescent signals of PCR amplification products, and carrying out result quantification and judgment analysis; When the fluorescent signal on the probe of the candida auriculata has a positive point, and the fluorescent signal on the probe of the internal reference gene has a positive point, the sample to be tested contains the candida auriculata; And when the fluorescent signal on the probe of the reference gene has no positive point, judging that the detection is abnormal, and retesting is needed. Preferably, the working concentrations of the different probes are respectively 150-300nM, the working concentrations of the upstream primers of the different primers are respectively 300-600nM, and the working concentrations of the downstream primers of the different primers are re