SLAMP primer group and kit for detecting candida albicans

Abstract

The invention provides an SLAMP primer group and a kit for detecting candida albicans, and relates to the technical field of biological detection. The SLAMP primer group is a primer group for amplification of a conserved region fragment of candida albicans, and a primer pair CA-SF / CA-SR has nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2. According to the method or the kit provided by the invention, rapid and accurate detection of the candida albicans can be completed without expensive instruments or complicated operation, so that the method or the kit is suitable for on-site rapid detection of ports, communities and other places with low professional degrees. According to the visual kit provided by the invention, great convenience is provided for field detection, and rapid and accurate non-diagnostic purpose detection of the candida albicans can be realized.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a SLAMP primer set and a kit for detecting Candida albicans. Background Art

[0002] Candida albicans is a fungus that usually exists in the oral cavity, upper respiratory tract, intestines and vagina of normal people. It is generally present in small numbers in normal bodies and does not cause disease. When the body's immune function or general defense power decreases or the mutual restraint of normal flora is disordered, the fungus reproduces in large quantities and changes its growth form (blastocyst phase) to invade cells and cause disease. Candida albicans exists as a commensal in a large part of the population and colonizes the oral cavity, gastrointestinal tract and reproductive tract asymptomatically. However, when the barrier integrity and / or the host immune response are disturbed, the fungus can migrate through the epithelium, invade the body's cells and cause disease.

[0003] At present, the commonly used detection methods for Candida albicans mainly include biochemical culture identification, serological detection, etc. The traditional detection method for Candida albicans takes about 5-10 days, and the detection limit is extremely low. It is time-consuming and labor-intensive. It cannot detect pathogenic bacteria in food in a timely and sensitive manner and cannot be used to deal with food emergencies. Compared with other detection methods for Candida albicans, the PCR method has excellent sensitivity, but it must undergo selective bacterial growth to dilute certain components that may interfere with the detection reaction. A complete PCR-based Candida albicans detection method takes 2 days to complete. It is urgent to establish a rapid, sensitive, and specific Candida albicans detection method. Summary of the invention

[0004] The purpose of the present application is to provide a SLAMP primer set for detecting Candida albicans, which has the advantages of high sensitivity and strong specificity.

[0005] Another object of the present application is to provide a kit for detecting Candida albicans, which has fast detection speed, high accuracy and is easy to use.

[0006] Another object of the present application is to provide a method for detecting Candida albicans, which is simple to operate.

[0007] In order to solve the above technical problems, the technical solution adopted in this application is:

[0008] In a first aspect, the present application provides a SLAMP primer set for detecting Candida albicans, wherein the SLAMP primer set is a primer pair CA-SF / CA-SR;

[0009] Among them, the nucleotide sequence of CA-SF is shown in SEQ ID NO.1, and the nucleotide sequence of CA-SR is shown in SEQ ID NO.2.

[0010] In a second aspect, the present application provides a kit, wherein the reaction system of the kit includes the above-mentioned SLAMP primer set.

[0011] In a third aspect, the present application provides a method for detecting Candida albicans using the above-mentioned kit, comprising the following steps:

[0012] Step 1, mixing the nucleic acid sample to be detected and the reaction system of the kit to prepare an amplification reaction solution;

[0013] Step 2, reacting the amplification reaction solution prepared above at 60-65° C. for 20-80 min, and judging whether the sample contains Candida albicans based on the color development result.

[0014] Short loop mediated isothermal amplification of nucleic acids (SLAMP) is a nucleic acid amplification technology under constant temperature conditions (Chinese patent application number: 202411655233.5) that was recently improved and developed by the inventor team based on the CDA technology (Chinese patent authorization number: ZL202110473121.8) and the Japanese LAMP nucleic acid amplification method developed in the early stage. This method mainly uses two different specific primers to identify the specific region of the target gene and perform amplification reactions under constant temperature conditions. Compared with conventional gene detection methods (such as PCR, etc.), the SLAMP reaction can be completed in a constant temperature water bath, with low instrumentation requirements and much simpler operation than traditional PCR and culture methods. It can be accurately completed without professionals, and the amplification results can be combined with the metal ion indicator hydroxynaphthol blue (HNB) to observe the color change by naked eye to realize the judgment of the results, making it suitable for primary medical institutions and local inspection and quarantine departments. Moreover, SLAMP can greatly shorten the operation time and reduce the chance of sample contamination, making it suitable for rapid diagnosis of Vibrio parahaemolyticus. In addition, the key circular primer used in SLAMP is about 30 bp, which is shorter than the 40 bp circular primer in LAMP. The design of the relevant primers is extremely simple, requiring only simple rearrangement, which will greatly save design time and development costs.

[0015] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0016] 1. The SLAMP primer set provided in the present application is designed and screened by the specific conserved region of Candida albicans (GenBank: MT193530.1), and consists of 2 primers, including an intra-sequence primer pair (CA-SF / CA-SR); the primer set provided by the present invention has high sensitivity and strong specificity, and the kit prepared therefrom can quickly and accurately detect Candida albicans contained in the sample to be tested.

[0017] 2. The primer set provided in this application has extremely high specificity, and the time required for SLAMP amplification is short, which further shortens the detection time and simplifies the operation.

[0018] 3. The kit provided by the present application and the use of the kit can complete the rapid and accurate detection of Candida albicans without expensive instruments or complicated operations, and therefore, is suitable for on-site rapid detection in airports, customs, ports, communities, etc. where the professional level is not high. The visualization kit provided by the present invention will greatly facilitate on-site detection and can achieve rapid and accurate detection of Candida albicans. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a curve diagram showing the change of fluorescence intensity with reaction time in Example 1.

[0021] Figure 2 This is a curve diagram showing the change of fluorescence intensity with reaction time in Example 2.

[0022] Figure 3 This is an endpoint monitoring chart based on color change of the reaction using HNB in ​​Example 3. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention is described in detail below in conjunction with the examples. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and can be specifically performed with reference to the specific methods listed in the book Molecular Cloning Laboratory Guide (3rd Edition) by J. Sambrook, or according to the kit and product instructions; the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0025] A SLAMP primer set for detecting Candida albicans, wherein the SLAMP primer set is a primer pair CA-SF / CA-SR;

[0026] The nucleotide sequence of CA-SF is shown in SEQ ID NO.1, and the nucleotide sequence of CA-SR is shown in SEQ ID NO.2. The specific nucleotide sequence is shown in Table 1:

[0027] Table 1 Primer sequences

[0028] Serial Number Primer name Nucleotide sequence SEQ ID NO.1 CA-SF CGTTCTTCATC-TTCTTCTTGGTTCTCGCATC SEQ ID NO.2 CA-SR GGAGGGCATGC-AAACGACGCTCAAACAG

[0029] A kit for detecting Candida albicans, wherein the reaction system of the kit comprises the above-mentioned SLAMP primer set.

[0030] In some embodiments of the present application, in the above SLAMP primer set, the concentrations of primer CA-SF and primer CA-SR are both 1-2 μM.

[0031] In some embodiments of the present application, the reaction system of the above-mentioned kit further includes Bst polymerase, SLAMP reaction buffer, reaction solvent and color developer.

[0032] In some embodiments of the present application, the developer is selected from one of Sybr green I, Eva green, hydroxynaphthol blue and chrome black T.

[0033] In some embodiments of the present application, the SLAMP reaction buffer comprises Tris-HCl, KCl, (NH 4 ) 2 SO 4 MgSO 4 and Triton X-100.

[0034] In some embodiments of the present application, the composition of the reaction system of the above-mentioned kit is:

[0035] 2~50mM Tris-HCl pH8.8, 2~20mM KCl, 2~20mM (NH 4 ) 2 SO 4 , 2~20mM MgSO 4, 0.1-0.5% Triton X-100, 0.2-1M betaine, 1-1.6mM dNTP, 5-10U Bst DNA polymerase, 100-150μmol / L color developer, 1-2μM primer CA-SF and 1-2μM primer CA-SR; the reaction solvent is ultrapure water.

[0036] A method for detecting Candida albicans using the above kit comprises the following steps:

[0037] Step 1, mixing the nucleic acid sample to be detected and the reaction system of the kit to prepare an amplification reaction solution;

[0038] Step 2, reacting the amplification reaction solution prepared above at 60-65° C. for 20-80 min, and judging whether the sample contains Candida albicans based on the color development result.

[0039] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0040] Example 1

[0041] In this example, Eva Green was used to verify the amplification reaction of the Candida albicans genomic DNA fragment.

[0042] Eva Green is similar to SYBR Green I. It is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helixes. Its inhibition on nucleic acid amplification reactions such as PCR is much less than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once it binds to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is related to the amount of double-stranded DNA, and the amount of double-stranded DNA in the nucleic acid amplification system can be detected based on the fluorescence signal.

[0043] The reaction solution was composed as follows (add ddH 2 (0 to 25 μL): 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 、14 mM MgSO 4 , 0.1% TritonX-100, 1M betaine, 1.25mM dNTP, 8U Bst DNA polymerase (NEW ENGLAND Biolabs), 1X Eva Green (Biotum), and primers: 1600nM CA-SF (shown in SEQ ID NO.1) and 1600nM CA-SR (shown in SEQ ID NO.2).

[0044] Target: Candida albicans genomic DNA dsDNA (shown in SEQ ID NO. 3);

[0045] A non-target control group was also set up.

[0046] The SLAN 96 real time PCR reaction temperature was set to 61°C and the reaction time was 60 min. The curve of fluorescence intensity versus reaction time is shown in Figure 1 shown. Figure 1 The results show that the primer set provided by the present invention can achieve rapid amplification of specific conserved regions in DNA extracted from Candida albicans, and the application of fluorescence detection therein can achieve the purpose of real-time monitoring, and the results can be judged by the real-time amplification curve.

[0047] Example 2

[0048] In this example, Eva Green was used to verify the amplification reaction of the Candida albicans SLAMP primer set on the genomes extracted from various bacteria, and the method was the same as that in Example 1.

[0049] The reaction solution was composed as follows (add ddH 2 (0 to 25 μL): 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 、14 mM MgSO 4 , 0.1% TritonX-100, 1M betaine, 1.25mM dNTP, 8U Bst DNA polymerase (NEW ENGLAND Biolabs), 1X Eva Green (Biotum), and primers: 1600nM CA-SF (shown in SEQ ID NO.1) and 1600nM CA-SR (shown in SEQ ID NO.2).

[0050] Target nucleic acid 1: genomic DNA extracted from Candida albicans (shown in SEQ ID NO.3), set as a positive control;

[0051] Target nucleic acid 2: genomic DNA extracted from Vibrio parahaemolyticus, set as negative control;

[0052] Target nucleic acid 3: Shigella extracted genomic DNA, set as negative control;

[0053] Target nucleic acid 4: Genomic DNA extracted from Klebsiella pneumoniae, set as negative control;

[0054] A non-target control group was also set up.

[0055] The SLAN 96 real time PCR reaction temperature was set to 61°C and the reaction time was 60 min. The curve of fluorescence intensity versus reaction time is shown in Figure 2 shown. Figure 2 The results showed that the primer set provided by the present invention can distinguish Candida albicans from Vibrio parahaemolyticus, Shigella and Klebsiella pneumoniae, which further illustrates the specificity of the SLAMP primer set provided by the present invention.

[0056] Example 3

[0057] In this example, hydroxynaphthol blue (HNB) was used to monitor the endpoint of the Candida albicans SLAMP amplification reaction.

[0058] Hydroxynaphthol blue (HNB) is a metal ion indicator that displays different indicator colors to judge the results according to the changes in the amount of magnesium ions or manganese ions combined with the by-product pyrophosphate in the reaction.

[0059] The composition of the reaction solution for SLAMP amplification of Candida albicans using hydroxynaphthol blue (HNB) is shown below.

[0060] The reaction solution was composed as follows, and ddH 2 O to 25 μL: 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 、14 mM MgSO 4 , 0.1% TritonX-100, 1 M betaine, 1.25 mM dNTP, 8 U Bst DNA polymerase (NEW ENGLAND Biolabs), 120 μM HNB and primers: 1600 nM CA-SF (shown in SEQ ID NO.1) and 1600 nM CA-SR (shown in SEQ ID NO.2).

[0061] Target nucleic acid: genomic DNA extracted from Candida albicans (shown in SEQ ID NO.3).

[0062] The amplification reaction was set up with 8 positive controls and 8 negative controls. The reaction temperature of the thermostatic water bath was set to 61°C and the reaction time was 60 min. Figure 3 As shown, violet indicates negative, and sky blue indicates positive. The experimental results show that the application of HNB can realize the judgment of reaction results by color, and the interpretation can be performed without the assistance of instruments.

[0063] In summary, the SLAMP primer set and kit for detecting Candida albicans in the embodiments of the present application have the following advantages: 1. The SLAMP primer set provided in the present application is obtained by designing and screening the specific conserved region of Candida albicans (GenBank: MT193530.1), and consists of 2 primers, including an intra-sequence primer pair (CA-SF / CA-SR). The primer set provided by the present invention has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect Candida albicans contained in the sample to be tested.

[0064] 2. The primer set provided in this application has extremely high specificity, and the time required for SLAMP amplification is short, which further shortens the detection time and simplifies the operation.

[0065] 3. The kit provided by the present application and the use of the kit can complete the rapid and accurate detection of Candida albicans without expensive instruments or complicated operations, and therefore is suitable for on-site rapid detection in airports, customs, ports, communities, etc. where the professional level is not high. The visualization kit provided by the present invention will greatly facilitate on-site detection and can achieve rapid and accurate detection of Candida albicans.

[0066] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. A SLAMP primer set for detecting Candida albicans, characterized in that: The SLAMP primer set is a primer pair CA-SF / CA-SR; Among them, the nucleotide sequence of CA-SF is shown in SEQ ID NO.1, and the nucleotide sequence of CA-SR is shown in SEQ ID NO.

2.

2. A kit for detecting Candida albicans, characterized in that: The reaction system of the kit comprises the SLAMP primer set according to claim 1.

3. A kit for detecting Candida albicans according to claim 2, characterized in that: In the SLAMP primer set, the concentrations of primer CA-SF and primer CA-SR are both 1 to 2 μM.

4. A kit for detecting Candida albicans according to claim 2, characterized in that: The reaction system of the kit also includes Bst polymerase, SLAMP reaction buffer, reaction solvent and color developer.

5. A kit for detecting Candida albicans according to claim 4, characterized in that: The developer is selected from one of Sybr green I, Eva green, hydroxynaphthol blue and chrome black T.

6. A kit for detecting Candida albicans according to claim 4, characterized in that: The SLAMP reaction buffer includes Tris-HCl, KCl, (NH4)2SO4, MgSO4 and Triton X-100.

7. A kit for detecting Candida albicans according to claim 2, characterized in that: The reaction system of the kit is composed of: 2~50mM Tris-HCl pH8.8, 2~20mM KCl, 2~20mM (NH4)2SO4, 2~20mM MgSO4, 0.1~0.5% Triton X-100, 0.2~1M betaine, 1~1.6mM dNTP, 5~10U Bst DNA polymerase, 100~150μmol / L color developer, 1~2μM primer CA-SF and 1~2μM primer CA-SR; the reaction solvent is ultrapure water.

Images