A fungal bacterial quadruple nucleic acid detection kit, a detection method and application thereof

Abstract

This invention discloses a quadruple nucleic acid detection kit for fungi and bacteria, its detection method, and its application. The kit comprises the following components: MIX reaction solution 1, MIX reaction solution 2, a positive control, and a negative control. MIX reaction solution 2 includes a first probe for detecting Candida albicans, a second probe for detecting Trichomonas vaginalis, a third probe for detecting Lactobacillus, and a fourth probe for detecting Megacoccus martensii. This kit utilizes multiplex quantitative PCR technology and designs specific probes and primers for Candida albicans, Trichomonas vaginalis, Lactobacillus, and Megacoccus martensii, enabling simultaneous detection of these fungi in the same reaction system. This overcomes the limitation of existing products that can only detect one virus or fungus in a single reaction tube.

Description

Technical Field

[0001] This invention relates to a fungal and bacterial quadruple nucleic acid detection kit, its detection method, and its application, belonging to the field of gene detection. Background Technology

[0002] Vulvovaginal candidiasis is a common gynecological disease. Candida albicans is a common pathogen, and about 75% of women will experience it at least once in their lifetime, with a recurrence rate of about 50%. Trichomonas vaginalis, a very common sexually transmitted disease, causes vaginitis and is prevalent worldwide. Trichomonas vaginalis is also a common pathogen causing infections of the human genitourinary system. As an anaerobic parasite that typically resides in the vagina and urinary tract, it often causes vaginitis and urethritis, and is a major risk factor for HIV infection and cervical cancer. Studies have shown that patients with cervical lesions have significantly reduced levels of vaginal lactobacilli, and lactobacilli have cytotoxic effects on cervical cancer cells. Dysbiosis of lactobacilli greatly increases the risk of cervical diseases. An increase in vaginal microorganisms such as macrococci can lead to lactobacillus dysbiosis, resulting in various cervical and vaginal diseases. Currently, the diagnosis of fungal and bacterial infections is mostly based on antiserological methods, but these methods have drawbacks such as low sensitivity and high false positive rates. In recent years, the use of fluorescent PCR technology for fungal detection has increased significantly. By designing TaqMan probes and primers targeting fungal specific sequences, the specificity of detection results can be largely guaranteed. However, existing kits for detecting Candida albicans, Trichomonas vaginalis, Lactobacillus, and Megacoccus maseri all employ individual detection methods, detecting only one virus or fungus in a single reaction tube. Furthermore, most existing kits suffer from poor specificity, low sensitivity, and low throughput, requiring further improvement. Summary of the Invention

[0003] The main objective of this invention is to provide a fungal and bacterial quadruple nucleic acid detection kit, which aims to solve the above-mentioned technical problems.

[0004] To achieve the above objectives, the present invention proposes a fungal and bacterial quadruple nucleic acid detection kit comprising the following components: MIX reaction solution 1, MIX reaction solution 2, a positive control, and a negative control. The MIX reaction solution 2 comprises a first probe for detecting Candida albicans, a second probe for detecting Trichomonas vaginalis, a third probe for detecting Lactobacillus, and a fourth probe for detecting Megacoccus maseri.

[0005] In one embodiment, the MIX reaction solution 2 includes a first component, a second component, a third component, and a fourth component. The first component includes a pair of first primers for detecting Candida albicans and a first probe for detecting Candida albicans. The second component includes a pair of second primers for detecting Trichomonas vaginalis and a second probe for detecting Trichomonas vaginalis. The third component includes a pair of third primers for detecting Lactobacillus and a third probe for detecting Lactobacillus. The fourth component includes a pair of fourth primers for detecting Megacoccus martensii and a fourth probe for detecting Megacoccus martensii.

[0006] In one embodiment, the sequence list is shown in the following table:

[0007]

[0008]

[0009] In one embodiment, the base sequence of the first primer is shown in SEQ ID NO.1 and SEQ ID NO.2, and the base sequence of the first probe is shown in SEQ ID NO.3.

[0010] In one embodiment, the base sequence of the second primer is shown in SEQ ID NO.4 and SEQ ID NO.5, and the base sequence of the second probe is shown in SEQ ID NO.6.

[0011] In one embodiment, the base sequence of the third primer is shown in SEQ ID NO.7 and SEQ ID NO.8, and the base sequence of the third probe is shown in SEQ ID NO.9.

[0012] In one embodiment, the base sequence of the fourth primer is shown in SEQ ID NO.10 and SEQ ID NO.11, and the base sequence of the fourth probe is shown in SEQ ID NO.12.

[0013] In one embodiment, the 5' ends of the first probe, the second probe, the third probe, and the fourth probe are all labeled with fluorescent reporter groups, and the 3' ends are all labeled with fluorescent quencher groups.

[0014] In one embodiment, the fluorescent reporter group includes any one of FAM, HEX, JOE, TET, CY3, CY5, VIC, and ROX, and the fluorescent quencher group includes any one of BHQ1, BHQ2, BHQ3, and Dabcy1. The fluorescent reporter groups in the first probe, second probe, third probe, and fourth probe are different.

[0015] In addition, the present invention also provides a detection method using the fungal and bacterial quadruple nucleic acid detection kit described above, the detection method comprising the following steps:

[0016] S1. Reagent preparation:

[0017] S11. Take out MIX reaction solution 1 from the kit, melt and mix it at room temperature, centrifuge briefly at low speed, take out MIX reaction solution 2 from the kit, centrifuge briefly at low speed, and place it on ice for later use.

[0018] S12. Determine the number of reactions N. The number of reactions N should be at least 2 more than the number of samples to be tested. Prepare the reaction system according to 10 μL × N of MIX reaction solution 1, 3 μL × N of reaction solution 2 and 4 μL × N of ddH2O.

[0019] S13. Shake the above mixture to mix well, centrifuge briefly for 5 seconds, and aliquot 18μL / tube into PCR tubes;

[0020] S2, Sample Preparation:

[0021] S21. Take 3 μL of the sample to be tested, the positive control, and the negative control, and add them separately to the aliquoted reaction mixture. Tightly cap the PCR reaction tube and centrifuge briefly at low speed. Then, run the PCR reaction tubes for analysis.

[0022] S3, PCR amplification detection:

[0023] S31. Set the loop parameters;

[0024] S32. Read the sample detection results based on the Ct value of the sample to be tested.

[0025] In addition, the present invention also provides an application of the fungal and bacterial quadrivalent nucleic acid detection kit as described above, the kit being used for in vitro detection of Candida albicans, Trichomonas vaginalis, Lactobacillus, and Megacoccus martensii.

[0026] The kit of this invention employs multiplex quantitative PCR technology, and designs specific probes and primers for Candida albicans, Trichomonas vaginalis, Lactobacillus, and Megacoccus martensii, enabling simultaneous detection of these bacteria in the same reaction system. The designed primers and probes are compared in the NCBI GeneBank database to verify their specificity.

[0027] This kit is easy to use and effectively prevents contamination. The PCR fluorescence detection time (from sample processing) is only 1.5-3 hours, and it can simultaneously detect Candida albicans, Trichomonas vaginalis, Lactobacillus, and Megacoccus martensii in the same reaction system. The PCR fluorescence detection is a completely closed process; after adding the sample extract and reaction solution, the tube cap does not need to be opened, reducing the chance of contamination.

[0028] This invention enables the simultaneous detection of Candida albicans, Trichomonas vaginalis, Lactobacillus, and Megacoccus martensii in the same reaction system, overcoming the limitation of existing products that can only detect one type of fungus in a single reaction tube. Furthermore, it offers advantages such as high sensitivity, strong repeatability, ease of operation, and rapid and objective results, making it highly promising for applications in clinical diagnosis and disease prevention and monitoring. Attached Figure Description

[0029] Figure 1 The kit used in this embodiment of the invention is shown in the amplification curves of 30 different vaginal microorganisms, including Candida albicans, Trichomonas vaginalis, Lactobacillus, and Megacoccus martensii. Detailed Implementation

[0030] The following detailed and complete description of the fungal and bacterial quadruple nucleic acid detection kit, its detection method, and its application provided by the present invention, with reference to specific embodiments, is provided. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available.

[0032] In this application, MIX reaction solution 1 includes Taq initiator enzyme, 10X buffer, 25 mM MgCl2, and 10 mM dNTPs. The positive control is a plasmid carrying the target gene fragment, and the negative control is sterile water.

[0033] Example 1: Preparation of the reagent kit

[0034] The sequence in this embodiment is as follows:

[0035]

[0036]

[0037] Among them, X1, X2, X3 and X4 are fluorescent reporter groups, and Y1, Y2, Y3 and Y4 are fluorescent quencher groups.

[0038] The test kit of this embodiment includes the following components (for 25 people):

[0039]

[0040] Example 2

[0041] 1. Reagent preparation

[0042] 1.1 Take out MIX reaction solution 1 from the kit, thaw and mix at room temperature, then centrifuge briefly at low speed. Take out MIX reaction solution 2 from the kit, centrifuge briefly at low speed, and place on ice for later use.

[0043] 1.2 Determine the number of reactions N: Calculate the required number of reactions based on the number of samples to be tested. If the number of samples is n, then the number of reactions N = (number of samples to be tested n + negative control 1 + positive control 1). Configure the reaction system according to the table below.

[0044] Reaction system configuration

[0045]

[0046] 1.3. Reaction system aliquoting: Prepare the appropriate number of PCR reaction tubes, vortex the above mixture to mix well, centrifuge briefly for 5 seconds, and aliquot 18μL / tube into PCR tubes.

[0047] 2. Sample preparation

[0048] 2.1 Samples were extracted using a commercially available DNA extraction kit. The specific procedures were performed according to the instructions for the nucleic acid extraction kit.

[0049] 2.2 Take 2 μL of the sample to be tested, the positive control, and the negative control, and add them separately to the aliquoted reaction mixture. Tightly cap the PCR reaction tube and centrifuge briefly at low speed. Analyze the PCR reaction tubes using the instrument.

[0050] 3. PCR amplification detection

[0051] 3.1 Place the PCR reaction tube in a real-time PCR instrument for amplification and detection. The instrument used in this invention is the Yarui MA6000 real-time PCR instrument.

[0052] 3.2. Loop Parameter Settings:

[0053]

[0054] 4. Determination of experimental validity

[0055] In this embodiment, the gene reporter fluorescence of Candida albicans is FAM, that of Trichomonas vaginalis is VIC, that of Lactobacillus is CY5, and that of Megacoccus masculinii is ROX.

[0056] (1) Positive control:

[0057] The FAM channel exhibits a typical S-shaped amplification curve and a Ct value ≤ 38.

[0058] The VIC channel exhibits a typical S-shaped amplification curve and a Ct value ≤ 38.

[0059] The CY5 channel exhibits a typical S-shaped amplification curve and a Ct value ≤ 38.

[0060] The ROX channel exhibits a typical S-shaped amplification curve and a Ct value ≤ 38.

[0061] (2) Negative control:

[0062] The Ct values ​​obtained from the FAM, VIC, CY5, and ROX channels are greater than or equal to 40 or have no Ct value. The lines are straight or slightly sloping and there is no exponential growth period.

[0063] (3) The experiment is valid only if conditions 1 and 2 are met simultaneously; otherwise, it is invalid.

[0064] 5. Judgment of test results

[0065] Taking ABI 7500 as an example: After the reaction is complete, the results are automatically saved. Adjust the start, end, and threshold values ​​of the baseline based on the analyzed image (users can adjust these values ​​according to their actual situation; the start value can be 3-15, and the end value can be 5-20. Set the threshold value in the Log graph window so that the threshold line is located in the exponential phase of the amplification curve, and the amplification curve of the negative control is flat or below the threshold line). Click Analysis to automatically obtain the analysis results, and read the detection results in the Report window.

[0066]

[0067]

[0068] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made under the concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A quadruple nucleic acid detection kit for fungi and bacteria, characterized in that, The kit comprises the following components: MIX reaction solution 1, MIX reaction solution 2, a positive control, and a negative control. MIX reaction solution 2 includes a first probe for detecting Candida albicans, a second probe for detecting Trichomonas vaginalis, a third probe for detecting Lactobacillus, and a fourth probe for detecting Megacoccus vallis. MIX reaction solution 2 includes a first component, a second component, a third component, and a fourth component. The first component includes a pair of first primers for detecting Candida albicans and a first probe for detecting Candida albicans. The second component includes a pair of second primers for detecting Trichomonas vaginalis and a second probe for detecting Trichomonas vaginalis. The third component includes a pair of third primers for detecting Lactobacillus and a third probe for detecting Lactobacillus. The fourth component includes a pair of fourth primers for detecting Megacoccus vallis and a fourth probe for detecting Megacoccus vallis. The base sequence of the first primer is shown in SEQ ID NO.1 and SEQ ID NO.2 of the sequence listing, and the base sequence of the first probe is shown in SEQ ID NO.3 of the sequence listing; The base sequence of the second primer is shown in SEQ ID NO.4 and SEQ ID NO.5 of the sequence listing, and the base sequence of the second probe is shown in SEQ ID NO.6 of the sequence listing; The base sequence of the third primer is shown in SEQ ID NO.7 and SEQ ID NO.8, and the base sequence of the third probe is shown in SEQ ID NO.

9. The base sequence of the fourth primer is shown in SEQ ID NO.10 and SEQ ID NO.11, and the base sequence of the fourth probe is shown in SEQ ID NO.

12.

2. The fungal and bacterial quadruple nucleic acid detection kit according to claim 1, characterized in that, The first probe, the second probe, the third probe, and the fourth probe are all labeled with fluorescent reporter groups at their 5' ends and fluorescent quencher groups at their 3' ends.

3. The fungal and bacterial quadruple nucleic acid detection kit according to claim 2, characterized in that, The fluorescent reporter group includes any one of FAM, HEX, JOE, TET, CY3, CY5, VIC, and ROX, and the fluorescent quencher group includes any one of BHQ1, BHQ2, BHQ3, and Dabcy1. The fluorescent reporter groups in the first probe, second probe, third probe, and fourth probe are different.

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