Oligonucleotide combination and kit for detecting and distinguishing cryptococcus

Abstract

The invention relates to an oligonucleotide combination and kit for detecting and distinguishing cryptococcus. The oligonucleotide combination comprises a specific primer, a guide chain and a reporter probe, wherein the specific primer, the guide chain and the reporter probe are designed aiming at a cryptococcus neoformans CAP59 gene and a cryptococcus gattii GPA1 gene. The kit comprises the oligonucleotide combination, pfAgo enzyme, divalent manganese ions and a reaction buffer solution, and can be used for non-diagnostic purpose detection. The method comprises the following steps: carrying out asymmetric PCR amplification on a sample DNA to obtain a single-chain product, incubating the single-chain product with pfAgo enzyme, a guide chain and a reporter probe together to carry out a cleavage reaction, and finally distinguishing the cryptococcus species through fluorescence signals (such as FAM and ROX). The method has the advantages of high specificity and sensitivity (the lower detection limit reaches 1CFU / mL), no cross reaction, short detection time (about 60 minutes), accurate verification in clinical cerebrospinal fluid samples, and suitableness for rapid identification of cryptococcus.

Description

Technical Field

[0001] This invention relates to the field of cryptococcal detection technology, and more particularly to an oligonucleotide combination and kit for detecting and differentiating cryptococci. Background Technology

[0002] Differentiating Cryptococcus neoformans ( Cryptococcus neoformans ) and Cryptococcus grete ( Cryptococcus gattii This not only addresses the needs of microbial taxonomy but also has crucial guiding significance for clinical diagnosis and treatment, directly impacting the identification of susceptible populations, the selection of treatment regimens, and the tracing of the source of infection. Among existing molecular diagnostic techniques, the TaqMan PCR probe method is widely used for the identification and detection of such pathogens. By designing specific probes, it can distinguish between two types of Cryptococcus. However, this method has limited distinguishing ability in cases of high sequence similarity or the presence of single nucleotide polymorphisms (SNPs), making it difficult to achieve high-precision single-base resolution.

[0003] In recent years, Argonaute (Ago) enzymes have been increasingly applied in the field of pathogen nucleic acid detection due to their ability to cleave specific nucleic acid sequences under guide strand (guide DNA / RNA) mediation. By designing specific guide strands to bind to target sequences and combining them with fluorescent reporter probes, the Ago system can achieve signal output and detection. Simultaneously, multiple signal detection can be performed in a single reaction, which is significantly different from other cascade cleavage reactions such as CRISPR. However, because Ago enzymes require a high amount of template, they are usually used in conjunction with nucleic acid amplification techniques, such as conventional PCR. In existing technologies, when PCR amplification products are directly used in subsequent Ago reactions, the two guide strands are often relied upon to cleave the target sequentially to generate a fluorescent signal. This process is slow and has low detection efficiency, limiting its application in rapid diagnostics.

[0004] Therefore, those skilled in the art need to develop an identification method that can balance high specificity, high sensitivity, and rapid detection capabilities. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an oligonucleotide combination and kit for detecting and differentiating Cryptococcus.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide an oligonucleotide combination for detecting and differentiating Cryptococcus, including: the oligonucleotide set for detecting Cryptococcus neoformans: a forward primer with a sequence as shown in SEQ ID NO: 1, a reverse primer with a sequence as shown in SEQ ID NO: 2, a guide strand with a sequence as shown in SEQ ID NO: 3, and a reporter probe with a sequence as shown in SEQ ID NO: 4; and / or the oligonucleotide set for detecting Cryptococcus gattii: a forward primer with a sequence as shown in SEQ ID NO: 5, a reverse primer with a sequence as shown in SEQ ID NO: 6, a guide strand with a sequence as shown in SEQ ID NO: 7, and a reporter probe with a sequence as shown in SEQ ID NO: 8.

[0007] Further, the reporter probe as shown in SEQ ID NO: 4 is labeled with a fluorescent reporter group FAM at its 5' end and a quencher group BHQ1 at its 3' end; The reporter probe as shown in SEQ ID NO: 8 is labeled with a fluorescent reporter group ROX at its 5' end and a quencher group BHQ2 at its 3' end.

[0008] Further, the guide strands as shown in SEQ ID NO: 3 and SEQ ID NO: 7 are phosphorylated at their 5' ends.

[0009] The second aspect is to provide an application of the oligonucleotide combination of the first aspect in a kit for detecting and distinguishing Cryptococcus.

[0010] Further, the Cryptococcus includes Cryptococcus neoformans and / or Cryptococcus gattii.

[0011] The third aspect is to provide a kit for detecting and distinguishing Cryptococcus, comprising the oligonucleotide combination of the first aspect.

[0012] Further, the kit further comprises pfAgo enzyme, divalent manganese ions, and reaction buffer.

[0013] Further, the kit further comprises a PCR reaction solution for performing asymmetric PCR amplification.

[0014] The fourth aspect is to provide a method for detecting and distinguishing Cryptococcus for non-diagnostic purposes, using the kit described above to detect the sample to be tested.

[0015] Further, the method comprises the following steps: S1, using the primers in the oligonucleotide combination to perform asymmetric PCR amplification on the sample DNA to obtain an amplified product rich in single-stranded DNA; S2, incubating the amplified product obtained in S1 with pfAgo enzyme, divalent manganese ions, and the guide strand and reporter probe in the oligonucleotide combination to perform a pfAgo cleavage reaction; S3, by detecting the fluorescence signal, to distinguish and identify the species of Cryptococcus.

[0016] Compared with the prior art, the above technical scheme has the following technical effects: The application realizes accurate differentiation of C. neoformans and C. gattii by designing specific primers and guide chains for the CAP59 gene of C. neoformans and the GPA1 gene of C. gattii, and combining the high-precision cutting characteristics of pfAgo enzyme, and has no cross-reaction with other common pathogens.

[0017] The application uses an asymmetric PCR and single-guide-chain-mediated pfAgo cutting strategy to push the lower limit of detection to 1 cfu / mL, significantly improving the detection capability of low-load samples, and providing a reliable tool for early diagnosis and occult infection.

[0018] The application uses asymmetric PCR to generate single-stranded targets, so that pfAgo can complete target recognition and report probe cutting with only one guide chain, and the detection time is controlled to about 60 minutes, overcoming the problems of slow reaction (about 90 minutes) and complex process caused by the dependence on double guide chains in traditional PCR-Ago combination.

[0019] In real cerebrospinal fluid sample verification, the detection results of the application are completely consistent with the clinical culture and mass spectrometric identification results, indicating that it still maintains high accuracy and stability in complex clinical samples, and has good clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The fluorescence signal-time curve for the sensitivity detection of C. neoformans.

[0021] Figure 2 The fluorescence signal-time curve for the sensitivity detection of C. gattii. DETAILED DESCRIPTION

[0022] The application will be further described below in conjunction with the drawings and specific embodiments, but not as a limitation of the application. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0023] The experimental methods in the following examples not specified in the specific conditions are usually according to the conventional conditions, or according to the conditions suggested by the manufacturer. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] 1.1 Design and sequence of primers and probes The CAP59 gene of C. neoformans (C. neoformans) and the GPA1 gene of C. gattii (C. gattii). Cryptococcus neoformans The CAP59 gene of C. neoformans (C. neoformans) and the GPA1 gene of C. gattii (C. gattii). Cryptococcus gattiiThe primers for asymmetric PCR, guide strand (gn) for pfAgo cleavage and fluorescent reporter probe were designed using Primer Express software for the GPA1 gene of C. noxigallicola, respectively. All sequence information is shown in Table 1 below. Table 1

[0025] 1.2 Sample processing and detection steps 1.2.1 Sample nucleic acid extraction: The sample DNA was extracted using Qiagen DNA tiq kit.

[0026] 1.2.2 First step: Asymmetric PCR amplification The PCR reaction system was prepared (total volume 20 μL) as follows: 2x PCR Mix (20 mM Tris-HCl (pH 8.3), 100 mM KCl, 3 mM MgCl2, 10% formamide, 0.4 mM dNTPs, 0.125 U / μL HotStart Taq DNA Polymerase): 10 μL Cnf-F2 (50 μM): 0.2 μL Cnf-R2 (1 μM): 0.2 μL Cgattii-F2 (50 μM): 0.2 μL Cgattii-R2 (1 μM): 0.2 μL Enzyme-free water: 0.2 μL DNA template: 9 μL PCR amplification program: 50 °C x 2 min→ 95 °C x 2 min→ (95 °C x 10 s + 60 °C x 15 s) x 45, total running time about 1 hour.

[0027] 1.2.3 Second step: pfAgo detection The pfAgo detection system was prepared (total volume 20 μL) as follows: PCR amplification product of the previous step: 5 μL pfAgo enzyme (10 μM): 1 μL pfAgo 10x Reaction Buffer (150 mM Tris / HCl, 2.5 M NaCl, pH 8.0): 2 μL MnCl2 (40 mM): 1 μL Cnf-pfAgo-gn (20 μM): 1 μL Cnf-pfAgo-RP (20 μM): 1 μL Cgattii-pfAgo-gn (20 μΜ): 1 μL Cgattii-pfAgo-RP (20 μΜ): 1 μL Enzyme-free water: 7 μL pfAgo cleavage reaction procedure: incubate the reaction system at 95 °C for 45 min, and collect the fluorescence signals of FAM channel (C. neoformans) and ROX channel (C. gattii) in real time during the process.

[0028] 1.3 Specificity test results To verify the specificity of the primer and probe set of the present application, the nucleic acids of various common pathogens were extracted and uniformly diluted to a concentration of 10 ^5 copies / μL, and then tested using the above method of the present example. The results are shown in Table 2 below.

[0029] Table 2

[0030] The experimental results show that the Cnf-F2 / R2, Cgattii-F2 / R2 primer set designed in the present application, and the guide chain Cnf-pfAgo-gn, Cgattii-pfAgo-gn and the report probe Cnf-pfAgo-RP, Cgattii-pfAgo-RP have very high specificity. The detection system can accurately and specifically distinguish C. neoformans and C. gattii, and has no cross reaction with other common pathogens listed.

[0031] 2.1 Purpose of the experiment To verify the amplification efficiency and sensitivity of the detection method of the present application for C. neoformans and C. gattii, and determine the dynamic range and detection lower limit of the detection method.

[0032] 2.2 Materials and methods 2.2.1 Experimental materials The primer and probe sequences listed in Table 1 in Example 1 were used.

[0033] 2.2.2 Sample preparation The DNA templates of C. neoformans and C. gattii were serially diluted, respectively: C. neoformans: 10 4 , 10 3 , 10 2 , 10 1 , 10 0 CFU / mL; C. gattii: 10 4 , 10 3 , 10 210 1 10 0 CFU / mL; A template-free control was set up as a negative control.

[0034] 2.2.3 Detection Method Asymmetric PCR amplification and pfAgo detection were performed using the same methods described in Examples 1, 2.2 and 1.2.3, with three replicates for each concentration.

[0035] 2.3 Results and Analysis 2.3.1 Sensitivity of Cryptococcus neoformans detection like Figure 1 As shown, Cryptococcus neoformans in 10 0 -10 4 Significant fluorescence signal growth was observed across the CFU / mL concentration range: High concentration template (10) 4 CFU / mL): The fluorescence signal begins to rise within 10 minutes and eventually reaches a high-intensity plateau; Low concentration template (10 0 CFU / mL): The fluorescence signal began to rise after 20 minutes, with a lower signal intensity but still significantly higher than the negative control. Negative control: The fluorescence signal remained at the baseline level throughout.

[0036] 2.3.2 Sensitivity for Cryptococcus grebindus detection like Figure 2 As shown, Cryptococcus grebindus in 10 0 -10 4 Significant fluorescence signal growth was observed across the CFU / mL concentration range: High concentration template (10) 4 CFU / mL): The fluorescence signal begins to rise rapidly within 5 minutes; Low concentration template (10 0 CFU / mL): The fluorescence signal began to rise slowly after 30 minutes; Negative control: No specific fluorescence signal was generated.

[0037] 2.4 Conclusion The detection method of this invention exhibits excellent amplification efficiency and sensitivity: The detection limit for Cryptococcus neoformans is 10. 0 CFU / mL The detection limit for Cryptococcus grebindotus is as low as 10. 0 CFU / mL.

[0038] Twelve cerebrospinal fluid samples provided by a hospital were collected, and all the samples were preliminarily screened by clinical culture. The samples with bacterial colonies were further identified by MALDI-TOF mass spectrometry as a reference standard.

[0039] DNA extraction was performed on each cerebrospinal fluid sample using a Qiagen nucleic acid extraction kit, and the operation steps were strictly performed according to the kit instructions. The established cryptococcus detection kit was used to perform experiments according to the asymmetric PCR and pfAgo detection process described in Example 1. Each sample was simultaneously detected for C. neoformans and C. gattii, and the fluorescence signals of the FAM and ROX channels were read respectively. The detection results are shown in Table 3 below: Table 3

[0040] It can be seen that in the 12 clinical cerebrospinal fluid samples involved in this experiment, the detection results of the present application are completely consistent with the clinical culture and MALDI-TOF mass spectrometry identification results. Among them, samples 1, 2, 6, 7 and 12 were correctly identified as C. neoformans infection; samples 3, 8, 9 and 11 were accurately identified as C. gattii infection; samples 4, 5 and 10 were all negative, which is consistent with the clinical results. No cross-reaction or non-specific signal was observed in all samples, proving that the method has good applicability and accuracy in real clinical scenarios.

[0041] The above is only the preferred embodiment of the present application, and does not limit the implementation and protection scope of the present application. For those skilled in the art, it should be realized that any equivalent replacement and obvious changes made by applying the content and drawings of the present application should be included in the protection scope of the present application.

Claims

1. An oligonucleotide combination for detecting and differentiating Cryptococcus, characterized by, The oligonucleotide set for detecting Cryptococcus neoformans comprises: a forward primer with the sequence as shown in SEQ ID NO: 1, a reverse primer with the sequence as shown in SEQ ID NO: 2, a guide strand with the sequence as shown in SEQ ID NO: 3, and a reporter probe with the sequence as shown in SEQ ID NO: 4; and / or The oligonucleotide set for detecting Cryptococcus gattii comprises: a forward primer with the sequence as shown in SEQ ID NO: 5, a reverse primer with the sequence as shown in SEQ ID NO: 6, a guide strand with the sequence as shown in SEQ ID NO: 7, and a reporter probe with the sequence as shown in SEQ ID NO:

8.

2. The oligonucleotide combination according to claim 1, wherein the reporter probe as shown in SEQ ID NO: 4 is labeled with a fluorescent reporter group FAM at the 5' end and a quencher group BHQ1 at the 3' end; the reporter probe as shown in SEQ ID NO: 8 is labeled with a fluorescent reporter group ROX at the 5' end and a quencher group BHQ2 at the 3' end. the guide strands as shown in SEQ ID NO: 3 and SEQ ID NO: 7 are phosphorylated at the 5' end.

3. The oligonucleotide combination according to claim 1 or 2, characterized in that, 4. Use of the oligonucleotide combination according to claim 1 or 2 in the preparation of a kit for detecting and distinguishing Cryptococcus. The Cryptococcus comprises Cryptococcus neoformans and / or Cryptococcus gattii.

5. Use according to claim 4, characterized in that, The oligonucleotide combination according to claim 1 or 2.

6. A kit for detecting and differentiating Cryptococcus, characterized by, The pfAgo enzyme, divalent manganese ions, and a reaction buffer are further included.

7. The kit of claim 6, wherein A PCR reaction solution for performing asymmetric PCR amplification is further included.

8. The kit of claim 6, wherein The kit according to any one of claims 6-8 is used to detect a sample to be tested.

9. A method of detecting and differentiating Cryptococcus for non-diagnostic purposes, characterized in that, The method comprises the following steps:

10. The method of claim 9, wherein, S1, performing asymmetric PCR amplification on the DNA of the sample using the primers in the oligonucleotide combination to obtain an amplified product rich in single-stranded DNA; S2, incubating the amplified product obtained in S1 with the pfAgo enzyme, divalent manganese ions, and the guide strand and reporter probe in the oligonucleotide combination to perform a pfAgo cleavage reaction; S3, distinguishing and identifying the species of Cryptococcus by detecting the fluorescent signal. ​

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