Real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer, probe and kit for detecting special drug-resistant pedigree of candida albicans

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a real-time fluorescent quantitative PCR (polymerase chain reaction) primer, a probe and a kit for detecting a special drug-resistant pedigree of candida albicans. The nucleotide sequence of the specific primer F is as shown in SEQ ID NO. 1; the nucleotide sequence of the specific primer R is as shown in SEQ ID NO. 2; the nucleotide sequence of a probe matched with the primer for use is as shown in SEQ ID No.3. The kit prepared by the invention ensures high specificity and high sensitivity in a PCR amplification process, effectively distinguishes drug-resistant mutant strains from sensitive strains, keeps efficient detection capability in a low-copy-number DNA template, reduces non-specific amplification and background signal interference, and improves the reliability and accuracy of a detection result.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and specifically relates to a real-time fluorescent quantitative PCR primer, a probe and a kit for detecting a special drug-resistant lineage of Candida albicans. Background Art

[0002] Candida albicans is a common human commensal fungus that colonizes the human skin surface and intestines. At the same time, Candida albicans is also one of the most common pathogens causing invasive fungal disease (IFD) and seriously endangers the patient's life. At present, the treatment drugs for IFD are quite limited. Azole antifungal drugs, such as fluconazole, itraconazole, voriconazole and posaconazole, are the first choice for treating Candida infections. However, the widespread use of broad-spectrum antibacterial drugs, chemotherapy drugs, glucocorticoids and immunosuppressants, the large-scale development of organ transplantation and the rising prevalence of immunodeficiency diseases have led to the increasing drug resistance of Candida albicans. Although Candida albicans is mainly considered an opportunistic pathogen in the human body, it may also play an important role in the agricultural environment, especially in animal husbandry. Candida albicans can cause infections in poultry and livestock, affecting their growth and development and even causing serious diseases. In addition, with the widespread use of antimicrobial drugs, Candida albicans and other fungi have gradually developed resistance to antifungal drugs, which further increases the difficulty and cost of treatment.

[0003] At present, most clinical methods for detecting drug resistance of Candida albicans are culture-based fungal drug sensitivity tests. However, the bacterial culture method has a long separation cycle, a cumbersome process, and is easily contaminated, which is not conducive to early rapid diagnosis. Therefore, designing a kit and a detection method that can quickly and sensitively detect the azole resistance gene of Candida albicans is an urgent problem to be solved in this field.

[0004] The reduced sensitivity of Candida albicans to azole drugs is usually associated with mutations in the ERG11 gene encoding lanosterol 14-α-demethylase (Erg11p). This gene is the main target of azole drugs. Studies have shown that some mutations in the ERG11 gene lead to amino acid substitutions that significantly affect the binding ability of azole drugs to target proteins, thereby reducing the therapeutic effect of the drugs.

[0005] Specific qPCR primers and probes are designed for the special drug-resistant lineage of Candida albicans, which can achieve rapid and accurate detection of drug-resistant strains. These primers and probes are based on drug-resistant related genes or mutation sites, and can directly identify drug-resistant strains in clinical samples, avoiding the cultivation step and shortening the detection time. The matching kit standardizes the detection process and facilitates its promotion and application in medical institutions at all levels.

[0006] In summary, the development of real-time fluorescence quantitative PCR primers, probes and kits for detecting special resistance lineages of Candida albicans is extremely necessary for timely identification of resistant strains, guiding rational clinical drug use, controlling the spread of resistance, and effectively monitoring and warning of drug-resistant fungal diseases in agricultural production. Summary of the invention

[0007] In view of the defects of the prior art, the purpose of the present invention is to provide a real-time fluorescence quantitative PCR primer, probe and kit for detecting the special drug resistance lineage of Candida albicans. The present invention first compares the ERG11 gene of all strains of the special drug resistance lineage Clade 1-R-α and other lineage strains, finds out the sequence of the special drug resistance lineage-specific base difference of Candida albicans, and uses this segment of sequence as the target sequence for real-time fluorescence PCR detection of the special drug resistance lineage of Candida albicans. Based on the target sequence, the special drug resistance lineage-specific amplification primers and probes of Candida albicans are designed, and a real-time fluorescence PCR detection system is established to effectively realize the rapid nucleic acid detection of the special drug resistance lineage of Candida albicans.

[0008] The technical effect of the present invention is achieved by the following technical scheme: a real-time fluorescent quantitative PCR primer for detecting the special drug-resistant lineage of Candida albicans, wherein the PCR primer is divided into a specific primer F and a specific primer R; Preferably, the nucleotide sequence of the specific primer F is as shown in SEQ ID NO.1; Preferably, the nucleotide sequence of the specific primer R is as shown in SEQ ID NO.2; The present invention also provides a probe used in conjunction with the primer, and the nucleotide sequence of the probe is shown in SEQ ID No.3.

[0009] The present invention also provides a kit for detecting the special drug-resistant lineage of Candida albicans, which includes real-time fluorescence quantitative PCR primers and probes; Preferably, the kit further comprises AceQ qPCR probe Master Mix, ROX, a DNA template and a DNA extraction solution; Preferably, the DNA extraction solution comprises Buffer A and Buffer B; Preferably, the composition of Buffer A includes 0.1-1wt% Triton X-100, 100mM NaCl and 20mM Tris-HCl at pH 7.5; Preferably, the composition of Buffer B includes 100 μg / mL proteinase K, 1 mM EDTA and 0.1 mg / mL BSA; The kit provided by the present invention uses the total DNA of the sample to be tested as a template, uses the specific primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2 and the probe shown in SEQ ID NO.3 to perform real-time fluorescent quantitative PCR, and determines the result according to the amplification curve and the fluorescent signal.

[0010] Specifically, in the above scheme, the reaction system of the fluorescent quantitative PCR is 25 μL: Specific primer F 10μM 2μL Downstream primer R 10μM 2μL Probe-P 10μM 1μL 2×AceQ qPCR probe Master Mix 10 μL 50×ROX 0.4 μL DNA template 1 μL Add ddH2O to make up the total volume to 25 μL.

[0011] Preferably, the fluorescent quantitative PCR amplification reaction program of the kit is: pre-denaturation at 95°C for 10 min, 1 cycle; denaturation at 95°C for 20 s, annealing at 60°C for 45 s, 45 cycles; Preferably, each time a sample is tested in the present invention, an NTC control (no template control), a NEG (negative control) and a POS control (positive control) must be set up. The three controls play a decisive role in the interpretation of the results: Effective amplification: NTC(-), NEG(-), POS(+); Invalid amplification: NTC(-), NEG(-), POS(-), reagent failure; Invalid amplification: NTC(-), NEG(+), POS(+), sample contamination; Invalid amplification: NTC(+), NEG(+), POS(+), system contamination; Only sample test results under the condition of effective control amplification can be trusted, otherwise the test needs to be repeated.

[0012] When the three controls are effectively amplified during the test, the sample result judgment criteria are as follows: CT value ≤38: positive result; CT value>40: negative result; 38<CT value<40: Repeat the test. If the CT value is lower than 40, it is considered positive amplification; if it is higher than 40, it is considered negative amplification.

[0013] The beneficial effects of the present invention are as follows: The present invention provides a real-time fluorescence quantitative PCR kit for detecting special drug-resistant lineages of Candida albicans. By targeting the key A114S and Y257H double amino acid substitution mutations in the Erg11p protein, highly specific upstream primers, downstream primers and matching probes are designed to ensure high specificity and high sensitivity in the PCR amplification process, effectively distinguish between drug-resistant mutant strains and sensitive strains, maintain high-efficiency detection capabilities in low-copy number DNA templates, reduce nonspecific amplification and background signal interference, and improve the reliability and accuracy of the detection results.

[0014] The kit prepared by the present invention adopts an optimized dual lysis buffer (Buffer A and Buffer B), through efficient lysis and purification steps, it is ensured that the Candida albicans cells are thoroughly lysed, the high-purity DNA is released to the maximum extent, and the residue of PCR inhibitors is reduced, thereby significantly improving the accuracy and sensitivity of real-time fluorescence quantitative PCR detection. At the same time, the optimized sample lysis, DNA extraction and PCR amplification steps enable the entire detection process to be completed in a short time, much faster than the several days required for traditional culture and drug sensitivity test; Real-time fluorescence quantitative PCR technology not only has higher detection efficiency and lower cost, but the standardization and batch production of the kit further reduce the unit detection cost, and is suitable for large-scale application in medical institutions and research institutions of various sizes. In addition, the kit is compatible with a variety of real-time fluorescence quantitative PCR platforms, supports simultaneous detection of multiple samples, greatly improves detection flux and efficiency, and meets high-throughput detection requirements; by reducing unnecessary surfactants and reused ingredients, optimizing the buffer formula, not only reducing production costs, but also simplifying the operating process, saving resources. At the same time, the reagent prepared by the present invention can detect extremely low concentrations of Candida albicans, can effectively realize environmental samples, and can be effectively used to monitor environmental samples in poultry and livestock farms to detect and control Candida albicans infection at an early stage. In addition, it can also effectively monitor the distribution of Candida albicans in soil and irrigation water sources, and can be effectively applied on a large scale.

[0015] In summary, the real-time fluorescence quantitative PCR kit of the present invention has rapid, accurate and efficient detection capabilities, significantly improves the sensitivity and specificity of detection, shortens the diagnosis time, improves the treatment effect, enhances the ability to monitor drug resistance, and achieves high cost-effectiveness and high-throughput detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a standard curve diagram of the amplification of the special drug-resistant lineage of Candida albicans by the fluorescent PCR system in Example 1 of the present invention; Figure 2 This is a sensitivity detection diagram of the fluorescent PCR system in Example 3 of the present invention; Figure 3 This is the sensitivity test amplification curve of Candida albicans in Example 4 of the present invention. DETAILED DESCRIPTION

[0018] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the claimed invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the invention belongs. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are purchased through conventional commercial channels.

[0019] Example 1: Design and screening of primers and probes (1) The length of the primer is generally 15-30 bp, and the commonly used length is 18-27 bp. However, it should not be longer than 38 bp, because too long will cause its extension temperature to be greater than 74°C, which is not suitable for Taq DNA polymerase reaction; (2) Primer amplification length. Generally, the target fragment amplification length is 100-200 bp. (3) The GC content of primers is generally 40%-60%, preferably 45-55%. Too high or too low GC content is not conducive to initiation reaction. The GC content and Tm value of upstream and downstream primers should be kept close; (4) The base at the 3' end of the primer is generally not A, as A has a relatively high priming efficiency at the wrong priming site. Do not have more than three consecutive bases at the 3' end of the primer, such as GGG or CCC; (5) The bases should be randomly distributed, and there should not be four consecutive bases complementary between the primers themselves or between primers; (6) Primers should be specific. After primer design is completed, use BLAST search to confirm primer specificity.

[0020] According to the above principles, the primer and probe design of relevant species is completed, specifically: The present invention is based on the characteristics of the Clade 1-R-α lineage, which has two key point mutations, A114S and Y257H, in the ERG11 gene of Candida albicans. According to these two mutation sites, specific primers and molecular beacon fluorescent probes for detecting the Clade1-R-α lineage of Candida albicans are designed to achieve accurate identification of this specific drug-resistant lineage. The screened primer and probe sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 (Table 1).

[0021] Table 1. Primer and probe sequences obtained by screening

[0022] Example 2: Fluorescence quantitative PCR amplification system and amplification conditions The 25 μl reaction system for real-time fluorescence quantitative PCR is: Specific primer F 10μM 2μL Downstream primer R 10μM 2μL Probe-P 10μM 1μL 2×AceQ qPCR probe Master Mix 10 μL 50×ROX 0.4 μL DNA template 1 μL Add ddH2O to make up the total volume to 25 μL.

[0023] AceQ qPCR probe Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd., catalog number: Q112-02 The reaction procedure of real-time fluorescence quantitative PCR was as follows: pre-denaturation at 95°C for 10 min, 1 cycle; denaturation at 95°C for 20 s, annealing at 60°C for 45 s, 45 cycles.

[0024] Each time a sample is tested, NTC control (no template control), NEG (negative control) and POS control (positive control) must be set up. These three controls play a decisive role in the interpretation of the results: Effective amplification: NTC(-), NEG(-), POS(+); Invalid amplification: NTC(-), NEG(-), POS(-), reagent failure; Invalid amplification: NTC(-), NEG(+), POS(+), sample contamination; Invalid amplification: NTC(+), NEG(+), POS(+), system contamination.

[0025] Only sample test results under the condition of effective control amplification can be trusted, otherwise the test needs to be repeated.

[0026] When the three controls are effectively amplified during the test, the sample result judgment criteria are as follows: CT value ≤38: positive result; CT value>40: negative result; 38<CT value<40: Repeat the test. If the CT value is lower than 40, it is considered positive amplification; if it is higher than 40, it is considered negative amplification.

[0027] Example 3: Preparation of standard curve of real-time fluorescence PCR system for special drug-resistant lineage of Candida albicans The genomic DNA of the special drug-resistant lineage of Candida albicans as a standard concentration template was diluted 10 times in 5 concentration gradients, a total of 6 concentration gradients, and amplified using the above detection system. The standard curve of the real-time fluorescence PCR system of the special drug-resistant lineage of Candida albicans was drawn based on the amplification results, which showed good detection sensitivity. The results are as follows Figure 1 shown.

[0028] The genomic DNA of the special drug-resistant lineage of Candida albicans with a standard concentration template was diluted 10 times in 5 concentration gradients in sequence and amplified with the above detection system. A blank control group was set up, that is, a reaction system without template DNA. After the PCR amplification was completed, the maximum fluorescence signal value of each concentration sample at the end of the amplification was extracted. The results are as follows Figure 2 shown.

[0029] Example 4: Evaluation of sensitivity and specificity of real-time fluorescence PCR detection system: 1. Sensitivity evaluation The real-time fluorescent PCR method using the primer probe of Example 1 of the present invention was used to dilute the template with the extracted nucleic acid stock solution in 10-fold ratios, and the plasmid concentration was adjusted to 10 6 The concentration of copies / μL was used as the mother solution, and 6 concentration gradients were set, namely S0, S1, S2, S3, S4, and S5. The above detection system was used for amplification. The results showed that the detection results of the 6 gradient templates were all positive, the CT value was linearly related to the dilution, the sensitivity was 100%, and the detection limit of the system was 10 copies. The results are as follows Figure 3 shown.

[0030] 2. Specificity evaluation The real-time fluorescent PCR method using the primer probe of Example 1 of the present invention was used to detect 20 other common fungi (including Aspergillus, Candida, Cryptococcus, sphaerotheca and Talaromyces marneffei) and the human genome (Table 1), with Candida albicans Clade 1-R-α lineage strains as positive controls. The results showed that except for the positive control, the amplification results of the other 20 non-Candida albicans Clade 1-R-α lineage strains were all negative. All the pathogen templates listed below are stored in the laboratory of the Institute of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention.

[0031] Table 2. Specific pathogen templates for real-time fluorescence PCR used to detect specific drug-resistant lineages of Candida albicans

[0032] Example 5 Evaluation of the detection capability of simulated specimens In order to evaluate the ability of this method to detect specific drug-resistant strains of Candida albicans in complex samples, 10 3 CFU / mL of bacterial liquid was used to construct simulated specimens (12 in total): sputum, nasopharyngeal swab, human urine, human feces, human blood, human vomit, tap water samples, ginkgo leaves, flour, sandy soil, banana puree, and juice; the results showed that the test results of the above 12 specimens were all positive for the special resistance lineage of Candida albicans, and the results are shown in Table 3-9 below.

[0033] Table 3. CT values ​​of sputum, nasopharyngeal swab, human urine, human feces, and human blood simulation samples

[0034] Table 4. Ct values ​​of banana puree and juice simulation samples

[0035] Table 5. Amplification Ct values ​​of simulated human vomitus samples

[0036] Table 6. Ct values ​​of flour simulation samples amplified

[0037] Table 7. Ct values ​​of tap water simulation samples amplification

[0038] Table 8. Ct values ​​of Ginkgo biloba leaf simulated sample amplification

[0039] Table 9. Amplification Ct values ​​of sandy soil simulation samples

[0040] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A real-time fluorescence quantitative PCR primer for detecting a special drug-resistant lineage of Candida albicans, characterized in that: The PCR primers are divided into a specific primer F and a specific primer R; the nucleotide sequence of the specific primer F is shown in SEQ ID NO.1; The nucleotide sequence of the specific primer R is shown in SEQ ID NO.

2.

2. A real-time fluorescent quantitative PCR probe for detecting a special drug-resistant lineage of Candida albicans, characterized in that: The nucleotide sequence of the probe is shown in SEQ ID No.

3.

3. A kit for detecting the special drug resistance spectrum of Candida albicans, characterized in that: The kit includes real-time fluorescence quantitative PCR primers, probes, AceQ qPCR probe Master Mix, ROX, a DNA template and a DNA extraction solution.

4. A kit for detecting the special drug resistance spectrum of Candida albicans according to claim 3, characterized in that: The composition of the buffer A includes 0.1-1wt% Triton X-100, 100mM NaCl and 20mM Tris-HCl at pH 7.5; the composition of the buffer B includes 100μg / mL proteinase K, 1mM EDTA and 0.1mg / mL BSA.

5. A kit for detecting the special drug resistance spectrum of Candida albicans according to claim 4, characterized in that: The total DNA of the sample to be tested is used as a template, and the specific primer pair and the probe are used to perform real-time fluorescence quantitative PCR, and the result is determined according to the amplification curve and the fluorescence signal.

6. A kit for detecting the special drug resistance spectrum of Candida albicans according to claim 5, characterized in that: The sample to be tested can also be sputum, nasopharyngeal swab, human urine, human feces, human blood, human vomit, irrigation water sample, ginkgo leaf, flour, sandy soil, banana puree, and fruit juice.

7. A kit for detecting the special drug resistance spectrum of Candida albicans according to claim 6, characterized in that: The reaction system of the fluorescent quantitative PCR is 25 μL: Specific primer F 10μM 2μL Downstream primer R 10μM 2μL Probe-P 10μM 1μL 2×AceQ qPCR probe Master Mix 10 μL 50×ROX 0.4 μL DNA template 1 μL Add ddH2O to make up the total volume to 25 μL.

8. A kit for detecting the special drug resistance spectrum of Candida albicans according to claim 7, characterized in that: The reaction procedure of the real-time fluorescence quantitative PCR was: pre-denaturation at 95° C. for 10 min, 1 cycle; denaturation at 95° C. for 20 s, annealing at 60° C. for 45 s, 45 cycles.

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