Primer probe combination and kit for detecting trichophyton, microsporum and epidermophyton

Abstract

The invention discloses a primer probe combination and a kit for detecting trichophyton, microsporum and epidermophyton. The primer probe combination comprises a primer pair and a probe for detecting trichophyton, microsporum and epidermophyton. The kit comprises a nucleic acid amplification reaction solution containing the primer probe combination, an enzyme mixed solution, a negative control and a positive control. The nucleic acid detection kit for the trichophyton genus, the microsporum genus and the epidermophyton genus, disclosed by the invention, can be used for simultaneously detecting nucleic acids of the trichophyton genus, the microsporum genus and the epidermophyton genus in the same reaction tube. In addition, the detection kit further has the advantages of being high in specificity, high in sensitivity, easy and convenient to operate, high in repeatability, rapid and objective in detection result and the like, and an effective and rapid technical means is provided for in-vitro detection of the trichophyton genus, the microspore genus and the epidermophyton genus.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro molecular detection of pathogenic microorganisms, in particular to a primer-probe combination and a kit for detecting Trichophyton, Microsporum and Epidermophyton, and belongs to the field of biotechnology. Background Art

[0002] Dermatomycoses are fungi that can cause infections of the skin, hair, and nails (called dermatophytosis or ringworm). They are superficial fungi. The three genera Trichophyton spp., Microsporum spp., and Epidermophyton spp. are the most common pathogens that cause ringworm.

[0003] According to literature reports, dermatophyte infection is one of the most common skin diseases and affects up to 20-25% of the population at any given time, and its recurrence rate is also very high. The reason for recurrence of the disease may be insufficient drug dosage or treatment failure caused by the insensitivity of the pathogenic fungus to the drug, or it may be caused by reinfection with different strains after cure or even simultaneous infection with multiple strains. Therefore, accurate identification of different dermatophyte species is of great significance for effective clinical treatment.

[0004] The traditional laboratory diagnosis method of dermatophytosis is still based on microscopic examination and culture. The microscopic examination method refers to the use of a microscope to examine the sample. This method has low sensitivity and poor specificity, and it is difficult to distinguish between different genera. The culture method refers to collecting samples and growing them in the laboratory. This method has a low positive rate and a long culture cycle of up to 3-4 weeks. It is not timely enough for early clinical diagnosis and has little diagnostic significance. Since skin dermatophytes are superficial infections, it is difficult to establish immunological methods based on the principles of antigen and antibody detection. In addition, due to the low sensitivity and high false positives of immunological methods, it is difficult to use them routinely.

[0005] Therefore, it is necessary to propose a dermatophyte detection kit with rapid detection, strong specificity and high sensitivity. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a kit capable of simultaneously detecting Trichophyton, Microsporum and Epidermophyton in a single reaction tube, and the kit has the advantages of rapid detection, strong specificity, high sensitivity and the like.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] In a first aspect, the present application provides a primer-probe combination for detecting Trichophyton, Microsporum and Epidermophyton, the primer-probe combination comprising:

[0009] A primer pair and a probe for detecting Trichophyton, wherein the base sequences of the primer pair are shown in SEQ ID No: 1 and SEQ ID No: 2 respectively; the base sequence of the probe is shown in SEQ ID No: 3, wherein the 5' end of SEQ ID No: 3 is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group;

[0010] A primer pair and a probe for detecting Microsporum, wherein the base sequences of the primer pair are shown in SEQ ID No: 4 and SEQ ID No: 5 respectively; the base sequence of the probe is shown in SEQ ID No: 6, wherein the 5' end of SEQ ID No: 6 is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group;

[0011] Primer pairs and probes for detecting Epidermophyton, wherein the base sequences of the primer pairs are shown in SEQ ID No: 7 and SEQ ID No: 8 respectively; the base sequence of the probe is shown in SEQ ID No: 9, wherein the 5' end of SEQ ID No: 9 is labeled with a fluorescent reporter group and the 3' end is labeled with a fluorescent quencher group.

[0012] As a preferred embodiment, the fluorescent reporter group is selected from FAM, VIC, ROX, Cy3 or Cy5 fluorescent reporter groups, and SEQ ID No: 3, SEQ ID No: 6, SEQ ID No: 9 are selected from different fluorescent reporter groups; the fluorescent quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, Dabcy1 and TAMRA, but it is required to be able to quench the fluorescent reporter group labeled on the same probe.

[0013] As a preferred embodiment, the primer-probe combination further comprises:

[0014] The endogenous internal standard primer pair is shown in SEQ ID NO: 10-11, and the internal standard probe is shown in SEQ ID NO: 12. Among them, the 5' end of SEQ ID No: 9 is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0015] As a preferred embodiment, the fluorescent reporter group is selected from FAM, VIC, ROX, Cy3 or Cy5 fluorescent reporter groups, and SEQ ID No: 3, SEQ ID No: 6, SEQ ID No: 9 and SEQ ID No: 12 are selected from different fluorescent reporter groups; the fluorescent quenching group is selected from any one of BHQ1, BHQ2, BHQ3, Dabcy1 and TAMRA, but it is required to be able to quench the fluorescent reporter group labeled on the same probe.

[0016]

[0017] In a second aspect, the present application provides a detection kit for Trichophyton, Microsporum and Epidermophyton, comprising the primer-probe combination described in the first aspect.

[0018] As a preferred embodiment, the detection kit includes a nucleic acid amplification reaction solution, an enzyme mixture, a negative control and a positive control;

[0019] Wherein, the nucleic acid amplification reaction solution includes the primer-probe combination and PCR reaction reagents described in the first aspect.

[0020] As a preferred embodiment, the concentrations of primers SEQ ID NO: 1-2, SEQ ID NO: 4-5, and SEQ ID NO: 7-8 in the nucleic acid amplification reaction solution are 0.4-0.6 μM, respectively; the concentrations of probes SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9 in the amplification reaction solution are 0.25-0.35 μM, respectively; and the concentrations of endogenous internal standard primers SEQ ID NO: 10-11 and probe SEQ ID NO: 12 in the detection kit in the amplification reaction solution are 0.15-0.25 μM, respectively.

[0021] As a preferred embodiment, the enzyme mixture comprises: DNA polymerase, UDG enzyme and RNase-free water.

[0022] As a preferred embodiment, the PCR reaction reagents include: PCR buffer, MgCl2, dATP, dTTP, dCTP, dGTP, dUTP, and RNase-free water.

[0023] As a preferred embodiment, the detection kit is combined with a rapid PCR amplification procedure as follows:

[0024]

[0025] As a preferred embodiment, the positive control is a mixture of four plasmid DNAs, wherein the four plasmid DNAs are plasmid DNAs for cloning specific targets of Trichophyton, Microsporum and Epidermophyton, and an internal standard plasmid DNA;

[0026] The negative control is RNase-free water.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The primers and probes provided by the present invention have good sensitivity and specificity. When applied to a detection kit, the primers and probes can be used to efficiently identify and detect three dermatophyte species in patients with dermatophyte infection at one time, and the operation is simple and time-consuming (30 minutes). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 To validate the results of the sensitivity test for Trichophyton spp.;

[0030] Figure 2 To verify the results of the sensitivity test for Microsporum spp.;

[0031] Figure 3 Validation results for sensitivity testing of Epidermophyton spp.;

[0032] Figure 4 Validation of results for Trichophyton, Microsporum, and Epidermophyton specific tests;

[0033] Figure 5 To verify the results of the repeatability test for Trichophyton spp.;

[0034] Figure 6 The results of the repeatability test for Microsporum were verified;

[0035] Figure 7 The results are verified by repeatability test of Epidermophyton spp. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0037] Unless otherwise specified, the reagents and biological materials used below are commercial products.

[0038] Example 1 Preparation of a kit for detecting Trichophyton, Microsporum and Epidermophyton

[0039] (1) Nucleic acid amplification reaction solution:

[0040] Prepare the nucleic acid amplification reaction solution according to Table 1.

[0041] Table 1 Composition of nucleic acid amplification reaction solution

[0042] Reagent name Add volume (μL) / 50 servings PCR buffer 100 <![CDATA[MgCl2]]> 3 100mM dATP 2.5 100mM dGTP 2.5 100mM dCTP 2.5 100mM dUTP 1.25 100mM dTTP 1.25 100 μM TRI-F 4 100 μM TRI-R 4 100 μM TRI-P 2 100 μM MIC-F 4 100 μM MIC-R 4 100 μM MIC-P 2 100 μM EPI-F 4 100 μM EPI-R 4 100μM EPI-P 2 100 μM RNP-F 4 100 μM RNP-R 4 100 μM RNP-P 2 RNase-free water 297 Total volume 450

[0043] (2) Enzyme mixture

[0044] Prepare the enzyme mixture according to Table 2.

[0045] Table 2 Preparation of enzyme mixture

[0046] Reagent name Add volume (μL) / 50 reactions Hot start DNA polymerase (5U / μL) 30 UDG enzyme (10U / μL) 1 Enzyme diluent 19 total 50

[0047] (3) Positive control

[0048] Prepare positive control according to Table 3

[0049] Table 3 Preparation of positive control

[0050] Reagent name Add volume (μL) / 50 reactions <![CDATA[Trichophyton plasmid (10 7 copies / mL)]]> 0.5 <![CDATA[Microsporum plasmid (10 7 copies / mL)]]> 0.5 <![CDATA[Epidermophyton plasmid (10 7 copies / mL)]]> 0.5 <![CDATA[Internal standard plasmid (10 7 copies / mL)]]> 0.5 DNA storage solution 48 total 50

[0051] (4) Negative control

[0052] RNase-free water.

[0053] Example 2 Method for using the detection kit for Trichophyton, Microsporum and Epidermophyton

[0054] The method for using the detection kit of the present invention comprises the following steps:

[0055] (1) Reagent preparation (reagent preparation area)

[0056] Take out the reagent kit, melt and shake at room temperature, and centrifuge at low speed for 10 seconds. When preparing the reaction solution, please mark each tube of reaction solution to avoid misinterpretation of the test results.

[0057] Preparation of PCR reaction solution: Calculate the number of reaction reagents required per person N (N = number of samples + 1 tube of positive control + 1 tube of negative control), and prepare the PCR reaction system for each person as shown in Table 4.

[0058] Table 4 PCR reaction solution configuration table

[0059] Reaction solution components Added amount (μL) / per serving Nucleic acid amplification reaction solution 5 Enzyme mixture 10 Total volume 15

[0060] Prepare the PCR reaction solution, mix it thoroughly and dispense it into PCR reaction tubes at 15 μL / tube.

[0061] (2) Sample processing (sample processing area)

[0062] Nucleic acid extraction: Take the sample to be tested and use a commercial nucleic acid extraction kit to extract nucleic acid according to the kit instructions.

[0063] Add samples: Add 5 μL of the processed sample nucleic acid, negative control, and positive control to the PCR reaction tubes prepared above, with a final volume of 20 μL / tube. Close the tube cap tightly and centrifuge at low speed.

[0064] (3) PCR amplification detection (nucleic acid amplification area)

[0065] Place the reaction tube into a fluorescent PCR amplification instrument and set the reaction program (Table 5) to perform amplification detection.

[0066] Table 5 Reaction program settings

[0067]

[0068]

[0069] Note: During the amplification step, fluorescence detection was performed at 60°C, and the detection channels were FAM, VIC, ROX, and CY5. ROX calibration was not selected for ABI series fluorescence PCR instruments, and None was selected for the quenching group.

[0070] (4) Results analysis

[0071] Adjust the start and end values ​​according to the analyzed image (it is recommended to set the start value at 3-15 and the end value at 5-20, and adjust the amplification curve of the negative control to be flat or below the threshold line), click Analyze, and view the results in the report interface.

[0072] (5) Interpretation of test results

[0073] Table 6 Quality Control

[0074]

[0075] Table 7 Results interpretation

[0076] Target Positive Negative Trichophyton FAM≤38 FAM>38 or Undet Microsporum VIC≤38 VIC>38 or Undet Epidermophyton ROX≤38 ROX>38 or Undet Internal reference CY5≤38 CY5>38 or Undet

[0077] Example 3 Sensitivity Verification of Trichophyton, Microsporum and Epidermophyton Detection Kits

[0078] The specific sequences of the three bacterial genera and the sequence of the internal reference gene RNase P were connected to the pUC57 vector to synthesize four plasmid standards. The plasmid construction was completed by Shanghai Diying Biotechnology Co., Ltd. The concentration was measured by ultraviolet spectrophotometer, and the copy number of the plasmid was calculated according to the length and concentration of each plasmid. The calculation formula is as follows:

[0079] (copies / μL) = [plasmid concentration (ng / μL) × 10 -9 ×6.02×10 23 ] / (number of bases × 660)

[0080] The copy number results are shown in Table 8:

[0081] Table 8 Plasmid copy number results

[0082]

[0083]

[0084] The quantified plasmids were diluted to 500 copies / mL, and the sensitivity test was performed using the plasmids at this concentration as templates. At least 8 wells were tested for each bacterial genus to determine the sensitivity of the three bacterial genus of this kit at this concentration. The amplification system was prepared according to Table 4, and the amplification conditions were set according to Table 5. The experimental results are shown in Table 9:

[0085] Table 9 Sensitivity verification results

[0086]

[0087] The results are as follows Figure 1-3 As shown in the figure, it can be seen that the positive detection rates of Trichophyton, Microsporum and Epidermophyton at a concentration gradient of 0.5 copies / μL are all 100%, so the detection sensitivity of Trichophyton, Microsporum and Epidermophyton in this kit is 500 copies / mL.

[0088] Example 4 Specificity verification of the detection kit for Trichophyton, Microsporum and Epidermophyton

[0089] The specificity of this kit was verified by taking genomic DNA of Candida albicans, Candida tropicalis, Candida glabrata, Aspergillus, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa and human genomic DNA at a concentration of not less than 10,000 copies / mL. The amplification system was prepared according to Table 4, and the amplification conditions were set according to Table 5. The experimental results are shown in Table 10:

[0090] Table 10 Specificity verification results

[0091]

[0092]

[0093] The results are as follows Figure 4As shown in the figure, it can be seen that this kit has good specificity and will not cross-react with common bacteria, fungal pathogens and human genomic DNA.

[0094] Example 5 Repeatability Verification of Trichophyton, Microsporum and Epidermophyton Detection Kits

[0095] The quantified plasmids were diluted to 20,000 copies / mL, and the plasmids at this concentration were used as templates for repeatability experiments. Two groups were tested in total, each group was repeated 4 times, and the CV value was calculated based on the experimental results. The amplification system was prepared according to Table 4, and the amplification conditions were set according to Table 5. The experimental results are shown in Table 11:

[0096] Table 11 Repeatability verification results

[0097]

[0098] The results are as follows Figure 5-7 As shown in the figure, it can be seen that the CV of the repeated detection Ct values ​​of Trichophyton, Microsporum and Epidermophyton under the concentration gradient of 20000 copies / mL all meet the index of ≤5%, so this kit has good repeatability and high stability.

[0099] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A primer-probe combination for detecting Trichophyton, Microsporum and Epidermophyton, characterized in that: The primer-probe combination includes: A primer pair and a probe for detecting Trichophyton, wherein the base sequences of the primer pair are shown in SEQ ID No: 1 and SEQ ID No: 2 respectively; the base sequence of the probe is shown in SEQ ID No: 3, wherein the 5′ end of SEQ ID No: 3 is labeled with a fluorescent reporter group, and the 3′ end is labeled with a fluorescent quencher group; A primer pair and a probe for detecting Microsporum, wherein the base sequences of the primer pair are shown in SEQ ID No:4 and SEQ ID No:5 respectively; the base sequence of the probe is shown in SEQ ID No:6, wherein the 5' end of SEQ ID No:6 is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; Primer pairs and probes for detecting Epidermophyton, wherein the base sequences of the primer pairs are shown in SEQ ID No:7 and SEQ ID No:8 respectively; the base sequence of the probe is shown in SEQ ID No:9, wherein the 5' end of SEQ ID No:9 is labeled with a fluorescent reporter group and the 3' end is labeled with a fluorescent quencher group.

2. The primer-probe combination for detecting Trichophyton, Microsporum and Epidermophyton as claimed in claim 1, characterized in that: Primer probe combinations also include: The endogenous internal standard primer pair is shown in SEQ ID NO: 10-11, and the internal standard probe is shown in SEQ ID NO: 12, wherein the 5' end of SEQ ID No: 12 is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

3. The primer-probe combination for detecting Trichophyton, Microsporum and Epidermophyton as claimed in claim 1 or 2, characterized in that: The fluorescent reporter group is selected from FAM, VIC, ROX, Cy3 or Cy5 fluorescent reporter groups, and SEQ ID No: 3, SEQ ID No: 6, SEQ ID No: 9 and SEQ ID No: 12 are labeled with different fluorescent reporter groups; the fluorescent quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, Dabcy1 and TAMRA, but it is required to be able to quench the fluorescent reporter group labeled on the same probe.

4. A detection kit for Trichophyton, Microsporum and Epidermophyton, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 3.

5. The detection kit according to claim 4, characterized in that Includes nucleic acid amplification reaction solution, enzyme mixture, negative control and positive control; The nucleic acid amplification reaction solution comprises the primer-probe combination according to any one of claims 1 to 3 and a PCR reaction reagent.

6. The detection kit according to claim 5, characterized in that The concentrations of primers SEQ ID NO: 1-2, SEQ ID NO: 4-5, and SEQ ID NO: 7-8 in the nucleic acid amplification reaction solution are 0.4-0.6 μM, respectively; the concentrations of probes SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9 in the amplification reaction solution are 0.25-0.35 μM, respectively; the concentrations of endogenous internal standard primers SEQ ID NO: 10-11 and probe SEQ ID NO: 12 in the detection kit in the amplification reaction solution are 0.15-0.25 μM, respectively.

7. The detection kit according to claim 5, characterized in that The enzyme mixture comprises: DNA polymerase, UDG enzyme and RNase-free water.

8. The detection kit according to claim 5, characterized in that The PCR reaction reagents include: PCR buffer, MgCl2, dATP, dTTP, dCTP, dGTP, dUTP, and RNase-free water.

9. The detection kit according to claim 5, characterized in that The rapid PCR amplification procedure is as follows:

10. The detection kit according to claim 5, characterized in that: The positive control is a mixture of four plasmid DNAs, wherein the four plasmid DNAs are plasmid DNAs for cloning specific targets of Trichophyton, Microsporum and Epidermophyton, and an internal standard plasmid DNA; The negative control is RNase-free water.

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