Application of trichophyton OG7443 gene in trichophyton identification, primer pair and trichophyton identification method

Abstract

The invention provides application of an OG7443 gene of trichophyton in identification of trichophyton, a primer pair and an identification method of trichophyton, and relates to the technical field of biology. Based on comparative analysis of genomes of trichophyton and other fungi, the specific marker gene OG7443 which only exists in trichophyton, has single copy and is free of repetitive sequence interference is screened out, and the gene can be used for identification of trichophyton. The primer provided by the invention is designed aiming at the OG7443 gene of trichophyton, has strong specificity and high sensitivity, can accurately distinguish mixed florae possibly existing in a clinical sample, improves the diagnosis accuracy of fungal infection, not only can accelerate the determination of pathogenic bacterium types, but also can improve the diagnosis accuracy of fungal infection. And important technical support can be provided for targeted antifungal treatment and public health prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of an OG7443 gene of Trichophyton in the identification of Trichophyton, a primer pair, and an identification method of Trichophyton. Background Art

[0002] Dermatophytes are divided into three genera: Trichophyton, Microsporum, and Epidermophyton. Currently, over 30 species of dermatophytes have been reported to cause human infection, with Trichophyton being the most common. Trichophyton has attracted widespread attention in recent years from both academic and clinical communities due to its high pathogenicity, strong infectivity, and increasing resistance to common antifungal drugs (such as terbinafine and some azoles). In some populations with resistant strains, inappropriate use of glucocorticoids or inadequate timely diagnosis can lead to prolonged illness and increased risk of recurrent infection, severely impacting public health.

[0003] Traditional identification methods, such as simple morphological observation, culture characteristics, or simplified PCR-RFLP techniques, are not ideal for accurately distinguishing Trichophyton from other closely related fungi. Even commercial fungal detection kits often lack specific probes or primers for Trichophyton, resulting in a lack of rapid, accurate, and cost-effective detection methods in clinical practice.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first object of the present invention is to provide an application of the OG7443 gene of Trichophyton in the identification of Trichophyton, so as to solve the above technical problems.

[0006] The second object of the present invention is to provide a primer pair.

[0007] The third object of the present invention is to provide a primer set.

[0008] A fourth object of the present invention is to provide use of the above primer pair or primer set in the identification of Trichophyton spp.

[0009] A fifth object of the present invention is to provide a kit for identifying Trichophyton spp.

[0010] A sixth object of the present invention is to provide a method for identifying Trichophyton.

[0011] In order to achieve the above objectives, the following technical solutions are adopted:

[0012] In a first aspect, the present invention provides the use of the OG7443 gene of Trichophyton in the identification of Trichophyton.

[0013] In a second aspect, the present invention provides a primer pair, comprising an upstream primer and a downstream primer;

[0014] The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.1;

[0015] The nucleic acid sequence of the downstream primer is shown in SEQ ID NO.2.

[0016] In a third aspect, the present invention provides a primer set comprising the primer pair and a primer for identifying fungi.

[0017] As a further technical solution, the primers for identifying fungi include an upstream primer and a downstream primer. The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.3; the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.4.

[0018] In a fourth aspect, the present invention provides use of the above primer pair or primer set in the identification of Trichophyton spp.

[0019] In a fifth aspect, the present invention provides a kit for identifying Trichophyton, comprising the above-mentioned primer pair or primer set.

[0020] As a further technical solution, the kit further includes a PCR reaction premix;

[0021] The PCR reaction premix includes DNA polymerase, Mg 2+ , dNTPs and buffer.

[0022] In a sixth aspect, the present invention provides a method for identifying Trichophyton, comprising: extracting DNA from a sample to be tested, then performing PCR amplification using the primer set, and identifying Trichophyton based on the amplified product.

[0023] As a further technical solution, Trichophyton spp. is identified based on the size or nucleic acid sequence of the amplified product.

[0024] As a further technical solution, the PCR amplification program includes: pre-denaturation: 95°C, 4 minutes; denaturation: 95°C, 30 seconds, annealing: 60°C, 30 seconds, extension: 72°C, 30 seconds, a total of 35 to 40 cycles; extension: 72°C, 7 minutes.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] Based on comparative analysis of the genomes of Trichophyton and other fungi, the present invention screens out a specific marker gene OG7443 that exists only in Trichophyton and has a single copy and no interference from repeated sequences. This gene can be used for the identification of Trichophyton.

[0027] The primers provided by the present invention are designed for the OG7443 gene of the genus Trichophyton. They have strong specificity and high sensitivity, can accurately distinguish mixed bacterial communities that may exist in clinical samples, and improve the accuracy of fungal infection diagnosis. They can not only speed up the identification of pathogenic bacteria, but also provide important technical support for targeted antifungal treatment and public health prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 Schematic diagram of the typical colony morphology of isolated Trichophyton strains cultured on Sabouraud's medium for 7 days;

[0030] Figure 2 Agarose gel diagram of multiplex PCR amplification products. DETAILED DESCRIPTION

[0031] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0032] In a first aspect, the present invention provides the use of the OG7443 gene of Trichophyton in the identification of Trichophyton.

[0033] Based on comparative analysis of the genomes of Trichophyton and other fungi, the present invention screens out a specific marker gene OG7443 that exists only in Trichophyton and has a single copy and no interference from repeated sequences. This gene can be used for the identification of Trichophyton.

[0034] In a second aspect, the present invention provides a primer pair, comprising an upstream primer and a downstream primer;

[0035] The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.1:

[0036] 5′-AGATCCGCCAAATGACGTTGT-3′ (SEQ ID NO. 1);

[0037] The nucleic acid sequence of the downstream primer is shown in SEQ ID NO.2:

[0038] 5'-TGCCTAGCGGCTCTTGAAA-3' (SEQ ID NO. 2).

[0039] The primers provided by the present invention are designed for the OG7443 gene of Trichophyton spp., and have strong specificity and high sensitivity. They can accurately distinguish mixed flora that may be present in clinical samples, thereby improving the accuracy of fungal infection diagnosis.

[0040] In a third aspect, the present invention provides a primer set comprising the primer pair and a primer for identifying fungi.

[0041] This primer set can accurately identify Trichophyton in the sample to be tested.

[0042] In some optional embodiments, the primers for identifying fungi include an upstream primer and a downstream primer, and the nucleic acid sequence of the upstream primer is shown in SEQ ID NO.3:

[0043] 5′-TCCGTAGGTGAACCTGCGG-3′ (SEQ ID NO. 3);

[0044] The nucleic acid sequence of the downstream primer is shown in SEQ ID NO.4:

[0045] 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO. 4).

[0046] In a fourth aspect, the present invention provides use of the above primer pair or primer set in the identification of Trichophyton spp.

[0047] The primer pair or primer set of the present invention has strong specificity and high sensitivity and can be used for the identification of Trichophyton spp.

[0048] In a fifth aspect, the present invention provides a kit for identifying Trichophyton, comprising the above-mentioned primer pair or primer set.

[0049] The kit comprises the primers provided by the present invention, so the kit can be used for the identification of Trichophyton.

[0050] In some optional embodiments, the kit further comprises a PCR reaction premix;

[0051] The PCR reaction premix includes but is not limited to DNA polymerase, Mg 2+, dNTPs and buffer.

[0052] In a sixth aspect, the present invention provides a method for identifying Trichophyton, comprising: extracting DNA from a sample to be tested, then performing PCR amplification using the primer set, and identifying Trichophyton based on the amplified product.

[0053] This identification method is simple to operate and can accurately identify Trichophyton within 24 hours.

[0054] In some alternative embodiments, Trichophyton spp. is identified based on the size or nucleic acid sequence of the amplified product.

[0055] The primer pairs used in the present invention amplify approximately 200 to 350 bp, and the primers used to identify fungi amplify approximately 639 bp. If bands appear at both 300 and 639 bp, the organism is considered Trichophyton. If only a band appears at 639 bp but not at 300 bp, Trichophyton is preliminarily ruled out.

[0056] In some optional embodiments, the PCR amplification program includes: pre-denaturation: 95°C, 4 minutes; denaturation: 95°C, 30 seconds, annealing: 60°C, 30 seconds, extension: 72°C, 30 seconds, a total of 35 to 40 cycles; extension: 72°C, 7 minutes.

[0057] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0058] Example 1: Identification and biological characteristics of Trichophyton

[0059] (1) Strain source

[0060] A fungus was isolated from a suspected clinical infection case (tinea pedis specimen from a patient).

[0061] (2) Colony morphology observation

[0062] After culturing the isolated strain in Sabouraud's medium (28°C, constant temperature) for 7 days, the colonies were light white and fluffy with neat or slightly radial edges ( Figure 1 (left image in );

[0063] When the culture dish is inverted, a slight yellow or light brown pigmentation can be seen on the back of the culture medium ( Figure 1 (right image in the figure);

[0064] Under the microscope, a large number of filamentous hyphae and several typical cylindrical microspores can be seen, most of which are evenly distributed.

[0065] (3) Molecular identification

[0066] Genomic DNA was extracted and the ITS region sequence was amplified using universal primers ITS1 and ITS4. The sequences were then compared with known sequences in the NCBI database. The similarity was over 99% and the isolates were clustered in the Trichophyton clade.

[0067] Combined with morphological and biochemical tests, it was finally confirmed to be Trichophyton spp.

[0068] Example 2: PCR detection method based on specific fragment design

[0069] (1) Target fragment screening

[0070] Based on the sequencing data of Example 1, a systematic comparison was performed with high-quality reference genomes of Trichophyton mentagrophytes, T. interdigitale, T. rubrum, T. tonsurans, T. simii, and T. schoenleinii. The specific process is as follows: 1. Orthogonal gene clustering. OrthoFinder 2 analysis was performed on the high-quality genome to obtain single-copy genes. 2. Species-specific initial screening. Several single-copy genes and insertion / deletion variant fragments that exist only in the genus Trichophyton were located. 3. Fragment amplification assessment. CDS length and internal gaps were counted.

[0071] Combined with the actual PCR design requirements, a position suitable for primer design and with sufficient sequence conservation (fragment A) was screened out.

[0072] (2) Primer design and detection system

[0073] Using Primer-BLAST or similar software, design a specific primer pair for fragment A (forward F1 / reverse R1, sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively). The product length is approximately 300 bp.

[0074] To ensure the effectiveness of the PCR system, a conventional ITS primer pair (sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, with an amplification product length of approximately 639 bp) was introduced as an internal control or broad-spectrum detection primer;

[0075] Two pairs of primers can be used for multiplex PCR amplification in the same reaction tube. The amplification procedure is as follows:

[0076] Pre-denaturation at 95°C for 4 min;

[0077] 95°C for 30 s / 60°C for 30 s / 72°C for 30 s, for a total of 35-40 cycles;

[0078] Extension at 72°C for 7 min.

[0079] The amplified product was detected by 1.5% agarose gel electrophoresis. If a double band of 300 bp and 639 bp appeared, it was identified as Trichophyton spp. If only a 639 bp band was present, it was identified as a closely related species or other dermatophytes.

[0080] (3) Specificity and sensitivity test

[0081] Multiple strains of Trichophyton (including T. indotineae, T. interdigitale, T. mentagrophytes, T. schoenleinii, T. rubrum, T. tonsurans, and T. simmi) and several common dermatophytes collected or preserved from home and abroad, for verification;

[0082] The results show that ( Figure 2 ): For strains confirmed to be Trichophyton spp. (1 to 35, 41, 80 to 82, 84, 85, 87, 91 to 95, n = 47), this method yielded double-band positive results (target band approximately 300 bp, ITS band approximately 600 bp). However, for other clearly identified Trichophyton or non-Trichophyton samples (n = 61), only a single ITS band (approximately 600 bp) was detected or no product was found.

[0083] When serially diluted Trichophyton DNA was used as a template, the lowest detectable fungal genomic DNA concentration was approximately 50 to 100 ng / μL, demonstrating good sensitivity.

[0084] This monitoring activity provides a basis for public health departments to formulate intervention measures and rational clinical drug use plans.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the OG7443 gene of Trichophyton in the identification of Trichophyton.

2. A primer pair, characterized in that: The primer pair includes an upstream primer and a downstream primer; The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.1; The nucleic acid sequence of the downstream primer is shown in SEQ ID NO.

2.

3. A primer set, characterized in that: The invention comprises the primer pair according to claim 2 and a primer for identifying fungi.

4. The primer set according to claim 3, characterized in that The primers for identifying fungi include an upstream primer and a downstream primer. The nucleic acid sequence of the upstream primer is shown as SEQ ID NO.3; the nucleic acid sequence of the downstream primer is shown as SEQ ID NO.

4.

5. Use of the primer pair according to claim 2 or the primer set according to claim 3 or 4 in the identification of Trichophyton.

6. A kit for identifying Trichophyton, characterized in that: The method comprises the primer pair according to claim 2 or the primer set according to claim 3 or 4.

7. The kit according to claim 6, characterized in that The kit also includes a PCR reaction premix; The PCR reaction premix includes DNA polymerase, Mg 2+ , dNTPs and buffer.

8. A method for identifying Trichophyton, characterized in that: include: Extract DNA from the sample to be tested, then perform PCR amplification using the primer set described in claim 3 or 4, and identify Trichophyton spp. based on the amplified product.

9. The identification method according to claim 8, characterized in that Trichophyton spp. is identified based on the size or nucleic acid sequence of the amplified product.

10. The identification method according to claim 8, characterized in that The PCR amplification program included: pre-denaturation: 95°C, 4 minutes; denaturation: 95°C, 30 seconds, annealing: 60°C, 30 seconds, extension: 72°C, 30 seconds, for a total of 35 to 40 cycles; extension: 72°C, 7 minutes.

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