Compositions of primer probe for detecting candida auris, PCR reaction solution, kit and application
By designing ITS-specific primers and probes for Candida auris and adding Bacillus subtilis as an internal standard, a multiplex PCR system was constructed, which solved the problems of high cost, cumbersome procedures, and long waiting time for results in existing technologies for detecting Candida auris, and achieved rapid detection with high sensitivity and high specificity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
- Filing Date
- 2026-05-26
- Publication Date
- 2026-07-10
AI Technical Summary
Existing technologies for detecting Candida auris suffer from high testing costs, cumbersome procedures, and long result waiting times, and they also struggle to quickly and accurately distinguish the infecting bacterial species.
A primer-probe composition targeting the ITS-specific region of Candida auris was designed, and exogenous internal standard Bacillus subtilis was added to construct a multiplex PCR system, which simplifies the gel electrophoresis verification steps of ordinary PCR and achieves high sensitivity and high specificity detection.
It enables rapid, simple, and accurate detection of Candida auris, reduces detection costs, simplifies result interpretation, and improves detection efficiency.
Smart Images
Figure CN122357780A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more particularly to primer and probe compositions, PCR reaction solutions, kits, and applications for detecting Candida auris. Background Technology
[0002] Candida auris is a newly emerging pathogenic fungus. In recent years, the detection of non-Candida albicans has increased significantly, and among these, Candida auris's resistance to most antifungal drugs makes it one of the more important species. Since its first report in Japan in 2009, Candida auris has spread to more than 40 countries across six continents (excluding Antarctica) in just over a decade. This is because Candida auris infection can occur in people of all ages, but is most common in the elderly. Although Candida auris was first discovered in the external auditory canal, it can also colonize the skin surface for extended periods. It can survive on the surfaces of medical equipment and facilities for up to two weeks and exhibits high resistance to common disinfectants, easily causing nosocomial transmission and outbreaks. The diagnosis and treatment of Candida auris is also difficult, mainly because it can quickly develop resistance to antifungal drugs, making it difficult to cure. In conclusion, these characteristics of Candida auris determine that if it spreads globally, it will inevitably pose a serious threat to global public health. WHO data shows that approximately 10% of children worldwide experience more than 10 respiratory viral infections each year, with one million children dying from pneumonia. Pneumonia has become the third leading cause of death for children under five in my country. Therefore, the etiological diagnosis of respiratory infections is increasingly important in clinical practice.
[0003] In existing technologies, ① CHROMagar™ medium is clinically available for identifying Candida auris, where colonies exhibit a blue halo, and 100% sensitivity and specificity can be achieved after 36 hours of incubation. Traditional yeast phenotyping can misdiagnose Candida auris as Rhodotorula rubrum or other Candida species. However, this method is time-consuming and results are not readily available. ② Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) is an ideal identification technique, rapidly identifying the species and genotype of strains and providing antifungal susceptibility testing. However, the specialized equipment is expensive and cannot be performed in most hospitals, requiring external testing by third parties, increasing processing time. ③ Molecular biology methods, such as polymerase chain reaction (PCR), can also effectively identify Candida auris. With the widespread use of fluorescent PCR instruments, most hospitals can now perform the tests themselves. PCR testing is quick, yielding results within hours. However, after amplifying DNA fragments using conventional PCR, verification via electrophoresis is required, which is cumbersome and results are delayed. Summary of the Invention
[0004] The purpose of this invention is to provide a primer-probe composition for detecting Candida auris. The primer-probe is designed for the ITS specific region of Candida auris to distinguish the infecting species in clinical samples. The composition has the characteristics of high sensitivity, high specificity and multi-target detection, which simplifies the cumbersome steps of gel electrophoresis verification required by ordinary PCR, and the results are simple and direct to interpret.
[0005] To achieve this objective, the present invention adopts the following technical solution: A primer-probe composition for detecting Candida auris, comprising: a Candida auris detection component and a Bacillus subtilis detection component; The Candida auris detection components include: upstream primer Cau-F, downstream primer Cau-R, and probe Cau-P; The sequence of the upstream primer Cau-F is 5'-CGTGATGTCTTCTCACCAATCT-3'; The sequence of the downstream primer Cau-R is 5'-GCGGGTAGTCCTACCTGAT-3'; The sequence of the probe Cau-P is 5'-TTTGTGAATGCAACGCCACCGCGA-3'; The Bacillus subtilis detection components include: upstream primer IC-F, downstream primer IC-R, and probe IC-P; The sequence of the upstream primer IC-F is 5'-CGCACTTCATTTAGGCGACG-3'; The sequence of the downstream primer IC-R is 5'-TGTTACATTACCAACCGGCA-3'; The sequence of the probe IC-P is 5'-GCGGAATGGAAGCGGTGGACCAAG-3'.
[0006] Optimally, both probe Cau-P and probe IC-P have a fluorescent group at their 5' end and a quenching group at their 3' end.
[0007] Optimally, the fluorescent group is FAM, ROX, Cy5, or HEX.
[0008] The quenching group is BHQ1 or BHQ2.
[0009] A PCR reaction solution for detecting Candida auris, comprising the above-described primer and probe composition for detecting Candida auris.
[0010] A kit for detecting Candida auris, comprising the aforementioned PCR reaction solution.
[0011] A method for detecting Candida auris for non-diagnostic and non-therapeutic purposes, comprising: using the above-mentioned primer and probe composition for detecting Candida auris; preparing a kit by lyophilizing the primer and probe composition into lyophilized pellets; extracting target nucleic acid from the Candida auris sample to be tested to obtain nucleic acid elution products; reconstituted the nucleic acid elution products into the lyophilized pellets for amplification, and performing closed amplification on a microfluidic chip.
[0012] The use of a primer-probe composition for detecting Candida auris in the preparation of products for detecting or assisting in the detection of Candida auris.
[0013] The use of a primer-probe composition for detecting Candida auris in the preparation of products for the identification or auxiliary identification of Candida auris.
[0014] The use of a primer probe composition for detecting Candida auris in the preparation of products for the diagnosis or auxiliary diagnosis of Candida auris infection or diseases caused by Candida auris infection.
[0015] The use of a primer probe composition for detecting Candida auris in the preparation of products for screening for Candida auris infection or diseases caused by Candida auris infection.
[0016] Compared with the prior art, one of the above technical solutions has the following beneficial effects: This invention proposes a primer-probe composition for detecting Candida auris. The primers and probes are designed specifically for the ITS region of Candida auris, enabling differentiation of infecting bacteria in clinical samples. Simultaneously, an exogenous internal standard is added to the detection system to ensure effective monitoring of the detection process. Furthermore, the composition achieves a limit of detection of 200 copies / mL for Candida auris in a PCR amplification system. The multiplex system composed of the Candida auris detection component and the Bacillus subtilis detection component shows no cross-reactivity with other pathogens causing the same symptoms. The composition features high sensitivity, high specificity, and multi-target detection, simplifying the cumbersome gel electrophoresis verification steps required for conventional PCR. The results are simple and direct to interpret, solving the problems of high cost, cumbersome procedures, and long result waiting times in existing Candida auris detection methods. Attached Figure Description
[0017] Figure 1 This is a schematic diagram of the primers and probes used in the detection of Candida auris. Detailed Implementation
[0018] To facilitate understanding of the present invention, a more comprehensive description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with techniques or conditions described in the literature in the art or according to product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.
[0019] like Figure 1 A primer-probe composition for detecting Candida auris, comprising: a Candida auris detection component and a Bacillus subtilis detection component; The Candida auris detection components include: upstream primer Cau-F, downstream primer Cau-R, and probe Cau-P; The sequence of the upstream primer Cau-F is 5'-CGTGATGTCTTCTCACCAATCT-3'; The sequence of the downstream primer Cau-R is 5'-GCGGGTAGTCCTACCTGAT-3'; The sequence of the probe Cau-P is 5'-TTTGTGAATGCAACGCCACCGCGA-3'; The Bacillus subtilis detection components include: upstream primer IC-F, downstream primer IC-R, and probe IC-P; The sequence of the upstream primer IC-F is 5'-CGCACTTCATTTAGGCGACG-3'; The sequence of the downstream primer IC-R is 5'-TGTTACATTACCAACCGGCA-3'; The sequence of the probe IC-P is 5'-GCGGAATGGAAGCGGTGGACCAAG-3'.
[0020] This invention proposes a primer-probe composition for detecting Candida auris. The primers and probes are designed specifically for the ITS region of Candida auris, enabling differentiation of infecting bacteria in clinical samples. Simultaneously, an exogenous internal standard is added to the detection system to ensure effective monitoring of the detection process. Furthermore, the composition achieves a limit of detection of 200 copies / mL for Candida auris in a PCR amplification system. The multiplex system composed of the Candida auris detection component and the Bacillus subtilis detection component shows no cross-reactivity with other pathogens causing the same symptoms. The composition features high sensitivity, high specificity, and multi-target detection, simplifying the cumbersome gel electrophoresis verification steps required for conventional PCR. The results are simple and direct to interpret, solving the problems of high cost, cumbersome procedures, and long waiting times in existing Candida auris detection methods.
[0021] This protocol uses Bacillus subtilis as an exogenous internal standard to participate in the entire process of nucleic acid extraction and PCR amplification, enabling monitoring of the entire process, such as the extraction and amplification stages. The reasons for choosing Bacillus subtilis are as follows: 1) Non-human / non-sample source: Bacillus subtilis is a bacterium that is ubiquitous in the environment but does not typically parasitize human fungal flora. 2) Distant species relationship: Bacillus subtilis belongs to the bacterial domain, while Candida auris belongs to the fungal domain; the two are evolutionarily distant. This significant difference ensures that the internal control primers / probes will not bind non-specifically to the genome of the target fungus. When designing multiplex PCR, it is easy to design systems that amplify only Bacillus subtilis-specific genes (such as 16S rDNA or specific functional genes), unaffected by high concentrations of Candida auris template, and vice versa. 3) Easy quantification of single-copy genes: The Bacillus subtilis genome has a clear structure, unlike some filamentous fungi or complex genomes with numerous repetitive sequences. Choosing a single copy of its housekeeping gene (such as gyrB, rpoB, or a specific 16S rDNA region) as a target ensures the stability of the internal control signal. 4) Easy to prepare quality control samples: Bacillus subtilis is easy to culture (low nutritional requirements, fast growth, no biosafety hazards, BSL-1 level), and high-concentration, high-purity bacterial suspensions or genomic DNA can be easily prepared for standardized preparation of internal control working solutions in kits. 5) No clinical interference: In respiratory, blood, or sterile body fluid samples, Bacillus subtilis (non-specific pathogenic strains) is usually not a key clinical focus. Therefore, even if it is present in trace amounts in the sample, it will not interfere with the judgment of the original state of the clinical sample, and its background signal is easily distinguishable from positive amplification. 6) Candida auris (fungus) has a thick and complex cell wall, making it a "difficult-to-lyse" microbial type in nucleic acid extraction. Bacillus subtilis, as a Gram-positive bacterium, has a cell wall containing multiple dense peptidoglycan structures, also making it a bacterium with greater difficulty in nucleic acid extraction.
[0022] Optimally, both probe Cau-P and probe IC-P have a fluorescent group at their 5' end and a quenching group at their 3' end.
[0023] Optimally, the fluorescent group is FAM, ROX, Cy5, or HEX.
[0024] The quenching group is BHQ1 or BHQ2.
[0025] In the fluorescent groups, FAM is 6-carboxyfluorescein (or 5-carboxyfluorescein); ROX is carboxy-X-rhodamine (abbreviated as rhodamine); Cy5 is anthocyanin 5 (or Cyanine 5); HEX is hexachlorofluorescein (or hexachloro-6-carboxyfluorescein); in the quenching groups, BHQ1 is black hole quencher-1 (or black hole quencher-1); BHQ2 is black hole quencher-2 (or black hole quencher-2).
[0026] A PCR reaction solution for detecting Candida auris, comprising the above-described primer and probe composition for detecting Candida auris.
[0027] This protocol prepares a PCR reaction solution by combining primers and probes, and includes, for example, DNA template, DNA polymerase, dNTPs, and buffer. Modern PCR reaction solutions often use premixed reagents (such as 2×Taq MasterMix), which combine dNTPs, buffer, and Mg²⁺. + Premixing with polymerase, as in the examples below, allows for the reaction to proceed only by adding template and primers, significantly reducing operational errors and the risk of contamination.
[0028] A kit for detecting Candida auris, comprising the aforementioned PCR reaction solution.
[0029] In addition to the PCR reaction solution, the kit includes optional positive and negative controls, nucleic acid extraction / lysis reagents, loading buffers, and other consumables, enabling standardized, ready-to-use testing. In commercial kits, Bacillus subtilis is typically pre-packaged in lysis buffer / magnetic bead buffer. Operators only need to add the Candida auris sample, eliminating volumetric errors associated with manual pipetting and spiked loading. The Bacillus subtilis culture can also be added manually separately.
[0030] A method for detecting *Candida auris* for non-diagnostic and non-therapeutic purposes includes using a primer-probe composition for detecting *Candida auris* as described above. The primer-probe composition is prepared into a kit in the form of lyophilized pellets. Target nucleic acid is extracted from the *Candida auris* sample to be tested, obtaining nucleic acid elution products. The nucleic acid elution products are reconstituted and amplified into lyophilized pellets, then amplified in a closed manner on a microfluidic chip. Generally, before extracting the target nucleic acid, a known amount of *Bacillus subtilis* is added, and the *Bacillus subtilis* and *Candida auris* are extracted simultaneously.
[0031] This method is based on real-time quantitative PCR and molecular biology methods to detect Candida auris. The composition has the characteristics of high sensitivity, high specificity and multi-target detection, which simplifies the cumbersome steps of ordinary PCR that require gel electrophoresis verification, and the results are easy and direct to interpret.
[0032] This protocol designs specific primers and probes targeting the ITS-specific sequence of Candida auris, which can be used for the universal detection of four branches of Candida auris, including but not limited to the detection of Clade I, II, III, and IV subtypes.
[0033] The use of a primer-probe composition for detecting Candida auris in the preparation of products for detecting or assisting in the detection of Candida auris.
[0034] The use of a primer-probe composition for detecting Candida auris in the preparation of products for the identification or auxiliary identification of Candida auris.
[0035] The use of a primer probe composition for detecting Candida auris in the preparation of products for the diagnosis or auxiliary diagnosis of Candida auris infection or diseases caused by Candida auris infection.
[0036] The use of a primer probe composition for detecting Candida auris in the preparation of products for screening for Candida auris infection or diseases caused by Candida auris infection.
[0037] Example: Step 1: In this embodiment, primers and probes were designed based on conserved regions of the four branches of Candida auris (Clade I, II, III, and IV) and specific regions of other bacterial species found on NCBI. The selected set of primers and probes is shown in Table 1 below:
[0038] Both probes Cau-P and IC-P have a fluorophore at the 5' end (FAM) and a quencher at the 3' end (BHQ1).
[0039] Step 2: Prepare the PCR reaction solution by adding primer and probe composition to 2×PCR premixed reagent (2×Taq MasterMix, commercially available), water, and DNA template. The specific reaction system is shown in Table 2 below:
[0040] Step 3: Perform the PCR reaction. The PCR reaction procedure is as follows: ① Pre-denaturation: 95℃, 5 minutes, 1 cycle; ② Denaturation: 95℃, 5 seconds; Annealing extension and fluorescence collection: 60℃, 30 seconds; 40 cycles, collect fluorescence; ③ Instrument cooling: 25℃, 60 seconds, 1 cycle.
[0041] Samples from three sources representing four branches of Candida auris (Clade I, II, III, and IV) were diluted to 2000, 1000, 500, 200, 100, and 50 copies / mL, respectively. Using the aforementioned PCR reaction solution, each concentration was tested 20 times. Ct values were recorded, and the detection rate was calculated. The concentration corresponding to a detection rate ≥95% was the limit of detection. The results are shown in Table 3-6 below.
[0042] Note: As verified by experiments and shown in Tables 3-6, the positive detection rate of samples within the detection concentration of the comprehensive target can reach over 95%. To ensure the stability and reliability of clinical testing, the detection limit concentration of this primer-probe combination is 200 copies / mL, so as to continuously meet the positive detection rate standard of ≥95% in practical applications.
[0043] Step 5: Cross-reactivity test; Using the PCR reaction solution and PCR reaction program in the above steps, amplify pathogens with similar clinical symptoms and the same infection location as the detection target. The specific pathogens tested are shown in Table 7 below.
[0044] Step 6: Using the PCR reaction solution and PCR reaction program from the above steps, amplify the pathogens listed in Table 5 above, and the detection results are shown in Table 8 below;
[0045] Note: The experiment detected "N / A" for pathogens, indicating no amplification signal was detected. This shows that although the PCR reaction system was targeting closely related species like Candida, it did not mistakenly identify them as Candida auris. Therefore, this primer-probe combination only recognizes its specific target pathogen and does not misidentify the listed bacteria or viruses, thus proving that there is no cross-reaction between this primer-probe combination and the aforementioned pathogens.
[0046] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims
1. A primer-probe composition for detecting Candida auris, characterized in that, include: Detection components for Candida auris and Bacillus subtilis; The Candida auris detection components include: upstream primer Cau-F, downstream primer Cau-R, and probe Cau-P; The sequence of the upstream primer Cau-F is 5'-CGTGATGTCTTCTCACCAATCT-3'; The sequence of the downstream primer Cau-R is 5'-GCGGGTAGTCCTACCTGAT-3'; The sequence of the probe Cau-P is 5'-TTTGTGAATGCAACGCCACCGCGA-3'; The Bacillus subtilis detection components include: upstream primer IC-F, downstream primer IC-R, and probe IC-P; The sequence of the upstream primer IC-F is 5'-CGCACTTCATTTAGGCGACG-3'; The sequence of the downstream primer IC-R is 5'-TGTTACATTACCAACCGGCA-3'; The sequence of the probe IC-P is 5'-GCGGAATGGAAGCGGTGGACCAAG-3'.
2. The primer and probe composition for detecting Candida auris according to claim 1, characterized in that, Both probes Cau-P and IC-P have a fluorescent group at their 5' end and a quenching group at their 3' end.
3. The primer and probe composition for detecting Candida auris according to claim 2, characterized in that, The fluorescent group is FAM, ROX, Cy5, or HEX; The quenching group is BHQ1 or BHQ2.
4. A PCR reaction solution for detecting Candida auris, characterized in that, The invention comprises a primer-probe composition for detecting Candida auris as described in any one of claims 1-3.
5. A kit for detecting Candida auris, characterized in that, Includes the PCR reaction solution as described in claim 4.
6. A method for detecting Candida auris for non-diagnostic and non-therapeutic purposes, using a primer-probe composition for detecting Candida auris as described in any one of claims 1-3, characterized in that, The primer and probe composition was prepared into a kit in the form of lyophilized pellets; the target nucleic acid was extracted from the Candida auris sample to be tested to obtain the nucleic acid elution product; The nucleic acid elution products were reconstituted and amplified into lyophilized pellets, which were then amplified in a closed manner on a microfluidic chip.
7. The use of the primer and probe composition for detecting Candida auris as described in any one of claims 1-3 in the preparation of products for detecting or assisting in the detection of Candida auris.
8. The use of the primer and probe composition for detecting Candida auris as described in any one of claims 1-3 in the preparation of products for the identification or auxiliary identification of Candida auris.
9. The use of the primer probe composition for detecting Candida auris according to any one of claims 1-3 in the preparation of products for the diagnosis or auxiliary diagnosis of Candida auris infection or diseases caused by Candida auris infection.
10. The use of the primer probe composition for detecting Candida auris according to any one of claims 1-3 in the preparation of products for screening for Candida auris infection or diseases caused by Candida auris infection.