Method for detecting sporotrichum globosum based on real-time fluorescent quantitative PCR (polymerase chain reaction) technology and application of method
By designing real-time fluorescence quantitative PCR technology of specific primer groups F3, R3 and probe 3, the rapid reliability problem of spherical spherical identification was solved, and high sensitivity and specificity detection was achieved, supporting the accurate diagnosis and treatment of spherical spherical spherical infection.
Patent Information
- Application Number
- CN202510744708.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-08-12
AI Technical Summary
The prior art is difficult to quickly and reliably identify or assist in the identification of spherical spore filaments, resulting in difficulty in selecting treatment plans.
Specific primer groups F3, R3 and probe 3 were designed for real-time fluorescence quantitative PCR technology, and whether spherical spore filaments are contained in the sample by detecting the amplification curve and cycling threshold (Ct value).
It has achieved rapid, sensitive and high specific detection of spherical spore filaments, with both accuracy and specificity of 100%, providing an effective means for the diagnosis and treatment of spherical spherical spherical infection.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for detecting sphaerocystis based on real-time fluorescence quantitative PCR technology and an application thereof. Background Art
[0002] Sporotrichosis is a disease caused by the dimorphic fungus Sporothrix complex ( Sporothrix complex ) causes a chronic inflammatory granulomatous infection that primarily affects the skin and subcutaneous tissue. Strains that can cause disease in humans worldwide include: Sporothrix sphaeroides ( Sporothrix globosa ), Sporothrix schenckii ( Sporothrix schenckii ), Sporothrix brasiliensis ( Sporothrix brasiliensis ), Sporothrix mexicana ( Sporothrix mexicana ) and Sporothrix leucoderma ( Sporothrix brasiliensis According to current research, the pathogenic strains causing sporotrichosis in Asia are Sporothrix schenckii and Sporothrix cocci. Studies have also revealed significant differences in pathogenicity, genotype, and drug susceptibility between the two. The current gold standard for diagnosing sporotrichosis is identification following fungal culture. Other studies have also provided serological testing methods, but these are currently rarely used clinically. The combined use of these methods can effectively diagnose sporotrichosis, but the process is lengthy and often fails to distinguish between different Sporothrix species, which is crucial for determining appropriate treatment options. Given these difficulties, a rapid, reliable, and species-specific diagnostic technique is clinically needed for diagnosing cocci.
[0003] Molecular biology techniques are increasingly being used for the detection and identification of pathogens. Real-time fluorescence quantitative PCR (qPCR) involves adding a fluorescent group to a conventional PCR reaction system, utilizing the accumulated fluorescent signal to monitor the PCR process in real time, and finally using a standard curve to quantitatively analyze unknown templates. qPCR offers the advantages of high accuracy, sensitivity, specificity, and rapid detection. It can directly detect trace amounts of pathogens in clinical specimens, making it an important auxiliary diagnostic tool in clinical practice. Summary of the Invention
[0004] The main problem to be solved by the present invention is how to identify or assist in identifying sphaerotheca.
[0005] In order to solve the above problems, the present invention provides a primer set for identifying or assisting in the identification of Sporothrix sphaeroides.
[0006] The primer set provided by the present invention for identifying or assisting in the identification of sphaeropodial sporothrix consists of primer F3, primer R3 and probe 3; The primer F3 is the following a1) or a2): a1) a single-stranded DNA molecule shown in Sequence 7 in the Sequence Listing; a2) a single-stranded DNA molecule having the same function as sequence 7 after one or more nucleotides are substituted and / or deleted and / or added; The primer R3 is the following a3) or a4): a3) a single-stranded DNA molecule shown in Sequence 8 in the sequence listing; a4) a single-stranded DNA molecule having the same function as sequence 8 after one or more nucleotides are substituted and / or deleted and / or added; The probe 3 is the following a5) or a6): a5) a single-stranded DNA molecule shown in Sequence 9 in the sequence listing; a6) A single-stranded DNA molecule having the same function as sequence 9 after one or more nucleotides are substituted and / or deleted and / or added.
[0007] Furthermore, the molar ratio of the primer F3, the primer R3 and the probe 3 is 2:2:1.
[0008] The present invention also provides the use of the primer set described above in any one of the following b1) to b8): b1) preparing a product for identifying or assisting in identifying whether the test bacterium is Sporothrix sphaeroides; b2) identifying or assisting in the identification of whether the test bacteria is sphaerotheca; b3) preparing products for diagnosing or assisting in diagnosing whether a subject is infected with Sporothrix cocci; b4) diagnosing or assisting in the diagnosis of whether the subject is infected with Sporothrix cocci; b5) preparing a product for detecting or assisting in detecting whether a sample to be tested contains Sporothrix sphaeroides; b6) detecting or assisting in detecting whether the sample to be tested contains sphaerotheca; b7) preparing products for the identification or assistance in the identification of Sporothrix cocci from other mycobacteria; b8) Differentiate or assist in differentiating Sporothrix cocci from other mycobacteria.
[0009] The present invention also provides a kit containing the primer set described above.
[0010] The present invention also provides the use of the above-mentioned kit in any one of the following b1) to b8): c1) Identifying or assisting in the identification of whether the test bacteria is sphaerotheca; c2) diagnose or assist in diagnosing whether the subject is infected with Sporothrix cocci; c3) detecting or assisting in detecting whether the sample to be tested contains sphaerotheca; c4) Differentiate or assist in differentiating Sporothrix cocci from other mycobacteria.
[0011] The present invention also provides a method for preparing the above-mentioned kit, which can be the following d1) or d2): d1) individually packaging each primer in the primer set described above; d2) Mixing the primers in the primer set described above according to the ratio.
[0012] In the above method, the primer F3, the primer R3 and the probe 3 in the primer set in d2) are mixed together in a molar ratio of 2:2:1.
[0013] The present invention also provides a method for identifying or assisting in identifying whether a test bacterium is a sphaeroides sporothrix, comprising the following steps: 1) Extract nucleic acid from the bacteria to be tested, and use the nucleic acid from the bacteria to be tested as a template and the primer set described above for real-time fluorescence quantitative PCR; 2) After the reaction is complete, determine whether the test bacteria is Coccidioides by using the amplification curve and CT value: If the test bacteria has an S-shaped amplification curve and the Ct value is less than or equal to 37, the test bacteria is or is a candidate for Coccidioides; if the Ct value is greater than 40, the test bacteria is not or is not a candidate for Coccidioides; samples with Ct values between 37 and 40 require repeated testing; if the Ct value is still less than 40 in the repeated test, it is determined to be Coccidioides; if the Ct value exceeds 40, the test bacteria is not or is not a candidate for Coccidioides.
[0014] The above method can be used to distinguish sphaerotheca from other strains. The other strains can be any of the following: Epidermophyton floccosum ( Epidermophyton floccosum ), Sporothrix schenckii ( Sporothrix schenckii ), Exophiala dermatitidis ( Exophiala dermatitidis ), Staphylococcus aureus ( Staphylococcus golden ), Exophiala jangii ( Exophiala jeanselmei) , Streptococcus pyogenes ( Streptococcus pyogenic ), Aspergillus fumigatus ( Aspergillus fumigatus ), Staphylococcus epidermidis ( Staphylococcus epidermis ), Aspergillus flavus ( Aspergillus flavus ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Aspergillus niger ( Aspergillus niger), Trichophyton mentagrophytes, Aspergillus terreus, Trichophyton rubrum, Aspergillus nidulans, Microsporum canis Microsporum canis ), Alternaria alternata ( Alternaria alternata ), Microsporum gypseum ( Microsporum gypseum ), Cladosporium carinii ( Cladophialophora carrionii ), Epidermophyton floccosum ( Epidermophyton floccosum ), Cladosporium pedrosense ( Fonsecaea pedrosoi ), Candida albicans ( Candida albicans ), Penicillium marneffei ( Penicillium marneffei ), Candida glabrata ( White glabrous ), Fusarium solani ( Fusarium solani ), Candida tropicalis ( Candida tropicalis ), Candida ( Candida parapsilosis ), Candida parapsilosis ( Candida parapsilosis ), Mycobacterium marinum ( Marine Mycobacterium ), Cryptococcus ( Cryptococcus neoformans ), Mycobacterium leprae ( Mycobacterium leprae ), Cryptococcus neoformans ( Cryptococcus neoformans ), Exophiala dermatitidis ( Exophiala dermatitidis ).
[0015] The present invention also provides a method for identifying or assisting in identifying whether a sample to be tested contains sphaeroides, comprising the following steps: 1) extracting nucleic acid from the sample to be tested, and performing real-time fluorescence quantitative PCR using the nucleic acid as a template and the primer set described above; 2) After the reaction is complete, the amplification curve and the cycle threshold (Ct value) are used as judgment indicators. The judgment criteria for sample results are as follows: if the sample to be tested has an S-shaped amplification curve and a Ct value less than or equal to 37, the sample to be tested contains coccidioides; if the Ct value is greater than 40, the sample to be tested does not contain coccidioides; samples with Ct values between 37 and 40 need to be tested again; if the Ct value is still less than 40 in the repeated test, the sample to be tested contains coccidioides; if the Ct value exceeds 40, the sample to be tested does not contain coccidioides.
[0016] Furthermore, the real-time fluorescence quantitative PCR reaction is carried out in a PCR system, and the contents of the reaction system and the primer-probe combination in the PCR system meet the following conditions: 1) The PCR reaction system consisted of: 12.5 μL of qPCR premix, 1 μL each of primer F3 and primer R3 (final concentration 10 μM), 0.5 μL of probe 3 (final concentration 10 μM), 2 μL of template DNA, and RNase-free ddH2O to 25 μL. 2) The PCR reaction conditions were as follows: 1 cycle of pre-denaturation at 95°C for 10 min; 50 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 15 s; and fluorescence signals were collected during the extension phase of each cycle.
[0017] The present invention uses the sphaeroides-specific genes CAL and ITS as target genes, and designs specific primers and probes therefor. Then, a sphaeroides standard strain (CBS 120340) was used as a positive control, 34 patients were selected, and their pathological specimens were used to isolate clinical isolates of sphaeroides and 30 common fungal and bacterial pathogenic strains in clinical practice as the experimental group. At the same time, a blank control group was set up, and the above samples were tested by fluorescence quantitative PCR technology. The results showed that: the skin tissue samples of the 34 patients infected with sphaeroides in the experimental group were all positive, with an accuracy of 100%; the remaining colonies and blank controls were all negative, with a specificity of 100%. The present invention successfully established a method for detecting sphaeroides using real-time fluorescence quantitative PCR technology, which provides an effective technical means for the early diagnosis and treatment of patients infected with sphaeroides, and plays an important role in the auxiliary diagnosis and efficacy monitoring of sphaeroides. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the detection curve of clinical specimens.
[0019] Figure 2 This is the gradient detection curve of the Sg (CAL1) and Sg (CAL2) probe primer combinations.
[0020] Figure 3 This is the gradient detection curve of the Sg (ITS) probe primer combination.
[0021] Figure 4 This is the standard curve of real-time fluorescence PCR. DETAILED DESCRIPTION
[0022] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.
[0023] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.
[0024] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.
[0025] The strains used in the specific experiments in the following examples are described in: Development and evaluation of a rapid diagnostic method for Sporothrix globosa in Asia using quantitative real-time PCR, BMC Infect Dis, 24, 824 (2024). The public may obtain the biological material from the applicant for use only in repeating the experiments of the present invention and may not be used for other purposes.
[0026] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.
[0027] The standard strain of Sporothrix sphaeroides in the following examples is a product of CBS, numbered CBS 120340.
[0028] Example 1: Primer set for rapid identification of sphaeroides and identification method thereof 1. Primer Set and Identification Method for Rapid Identification of Sporothrix sphaeroides In the present invention, the Sporothrix sphaeroides-specific genes CAL (Genbank No. KP101477.1, updated on October 24, 2014; KC693877.1, updated on February 28, 2013) and ITS (Genbank No. KY387687.1, updated on December 16, 2016) were used as target genes. Specific primers and probes were designed using Beacon Designer 8.0 software. The specific sequences are shown in Table 1.
[0029] Table 1. Specific primers and probes
[0030] Note: Probe 1 carries a FAM-MGB fluorescent label, probe 2 carries a FAM-MGB fluorescent label, and probe 3 carries a FAM-MGB fluorescent label.
[0031] 2. qPCR method for rapid identification of sphaeroides DNA from Sporothrix sphaeroides was extracted using the Quick-DNA Fungal / Bacterial Miniprep Kit (D6005, Zymo Research, USA), primers and probes (Invitrogen), and TaqMan Gene Expression Master Mix (Thermo Fisher). The specific experimental steps are as follows: (1) Extract DNA according to the instructions of the Quick-DNA Fungal / Bacterial Miniprep Kit (D6005, ZymoResearch, USA). Use a spectrophotometer to measure the A260 / A280 ratio of DNA and ensure that it is between 1.8 and 2.0 to avoid protein or RNA contamination.
[0032] (2) Prepare 25 μL of PCR system according to the formula in Table 2. The final dosages of upstream primer F, downstream primer R, and probe in the PCR system are 1 μL, 1 μL, and 0.5 μL, respectively.
[0033] Negative and positive controls were set up at the same time, the negative control was ddH2O, and the positive control was a standard strain of Sporothrix sphaeroides (CBS 120340).
[0034] (3) The detection was performed using the American ABI StepOnePlus fluorescence quantitative PCR instrument. The reaction procedure was 95°C pre-denaturation for 10 min, 1 cycle; 95°C denaturation for 15 s, 60°C annealing for 15 s, 50 cycles.
[0035] Table 2. Reaction system
[0036] Result determination: The cycle threshold (Ct value) is used as the judgment indicator. The judgment criteria for sample results are: 1) If the Ct value is less than or equal to 37, the sample is judged to be positive.
[0037] 2) If the Ct value is greater than 40, the sample is judged to be negative.
[0038] 3) If the Ct value of the sample is between 37 and 40, it needs to be tested again. If the Ct value is still lower than 40 in the repeated test, the sample is judged as positive amplification, and if it exceeds 40, it is judged as negative amplification.
[0039] The reliability of the method was verified using isolates from 6 patients with sphaeroidal spore-trichosis. The average Ct value of the primer-probe combination for CAL1 was 20.84±2.99, the average Ct value of the primer-probe combination for CAL2 was 20.55±1.93, and the average Ct value of the primer-probe combination for ITS was 22.40±4.0 ( Figure 1 ).
[0040] Example 2: Sensitivity and specificity testing of the identification method for identifying sphaerocystis 1. Sensitivity test The genomic DNA solution of the standard strain of Sphaerotheca sphaeroides (CBS 120340) was serially diluted to obtain Sphaerotheca sphaeroides DNA solutions with concentrations of 6 ng / μl, 600 pg / μl, 60 pg / μl, 6 pg / μl, 600 fg / μl, 60 fg / μl, 6 fg / μl, and 0.6 fg / μl, respectively.
[0041] Using Sporothrix sphaeroides DNA solutions of different concentrations as templates, fluorescence quantitative PCR and result determination were performed using the method in step 2 of Example 1 and the specific combination of F3 / R3 / probe 3.
[0042] The results showed that the highest sensitivity of the detection method established by the present invention could reach 6 fg / μl.
[0043] Among them, the sensitivity of the primer-probe combination of CAL1 reached 60 fg / μl ( Figure 2 ), the sensitivity of the primer-probe combination of CAL2 reached 60 fg / μl ( Figure 2 ), the sensitivity of the ITS primer-probe combination reached 6 fg / μl ( Figure 3 ).
[0044] 2. Specificity test Specific experimental materials: Cultured colonies of skin tissue samples infected with common bacteria or fungi. As shown below: Epidermophyton floccosum, Sporothrix schenckii, Exophiala dermatitidis, Staphylococcus aureus, Exophiala jeanselmei, Streptococcus pyogenes, Aspergillus fumigatus, Staphylococcus epidermidis, Aspergillus flavus, Pseudomonas aeruginosa, Aspergillus niger, Trichophyton mentagrophytes, Aspergillus terreus, Trichophyton rubrum, Aspergillus nidulans, nidulans), Microsporum canis, Alternaria alternata, Microsporum gypseum, Cladophialophora carrionii, Epidermophyton floccosum, Fonsecaea pedrosoi, Candida albicans, Penicillium marneffei, Candida glabrata, Fusarium solani, Candida tropicalis, Candida parapsilosis, Candida parapsilosis, Mycobacterium marinum, Cryptococcus neoformans, Mycobacterium leprae, Cryptococcus neoformans neoformans), Exophiala dermatitidis. All patients were clinically diagnosed and provided informed consent.
[0045] Genomic DNA was extracted from each of the above experimental materials. The extracted DNA solution was used as a template. Fluorescence quantitative PCR and result determination were performed using the F / R / probe specific combination as described in step 2 of Example 1. ddH2O was used as a blank control.
[0046] The results showed that the primer-probe combination of CAL1, CAL2, and ITS detected 6 colonies of Sporothrix sphaeroides, with an accuracy of 100%, while the remaining colonies and the blank control were all negative, with a specificity of 100% ( Figure 4 ).
[0047] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.
Claims
1. A primer set for identifying or assisting in the identification of Sporothrix sphaeroides, consisting of primer F3, primer R3, and probe 3; The primer F3 is the following a1) or a2): a1) a single-stranded DNA molecule shown in Sequence 7 in the Sequence Listing; a2) a single-stranded DNA molecule having the same function as sequence 7 after one or more nucleotides are substituted and / or deleted and / or added; The primer R3 is the following a3) or a4): a3) a single-stranded DNA molecule shown in Sequence 8 in the sequence listing; a4) a single-stranded DNA molecule having the same function as sequence 8 after one or more nucleotides are substituted and / or deleted and / or added; The probe 3 is the following a5) or a6): a5) a single-stranded DNA molecule shown in Sequence 9 in the sequence listing; a6) A single-stranded DNA molecule having the same function as sequence 9 after one or more nucleotides are substituted and / or deleted and / or added.
2. The primer set according to claim 1, wherein: The molar ratio of the primer 1, the primer 2 and the probe is 2:2:
1.
3. Use of the primer set according to claim 1 or 2 in any one of the following b1) to b8): b1) preparing a product for identifying or assisting in identifying whether the test bacterium is Sporothrix sphaeroides; b2) identifying or assisting in the identification of whether the test bacteria is sphaerotheca; b3) preparing products for diagnosing or assisting in diagnosing whether a subject is infected with Sporothrix cocci; b4) diagnosing or assisting in the diagnosis of whether the subject is infected with Sporothrix cocci; b5) preparing a product for detecting or assisting in detecting whether a sample to be tested contains Sporothrix sphaeroides; b6) detecting or assisting in detecting whether the sample to be tested contains sphaerotheca; b7) preparing products for the identification or assistance in the identification of Sporothrix cocci from other mycobacteria; b8) Differentiate or assist in differentiating Sporothrix cocci from other mycobacteria.
4. A kit comprising the primer set according to claim 1 or 2.
5. Use of the kit according to claim 4 in any one of the following b1) to b8): c1) Identifying or assisting in the identification of whether the test bacteria is sphaerotheca; c2) diagnose or assist in diagnosing whether the subject is infected with Sporothrix cocci; c3) detecting or assisting in detecting whether the sample to be tested contains sphaerotheca; c4) Differentiate or assist in differentiating Sporothrix cocci from other mycobacteria.
6. The method for preparing the kit according to claim 4, comprising the steps d1) or d2) below: d1) individually packaging each primer in the primer set of claim 1 or 2; d2) mixing the primers in the primer set according to claim 1 or 2 in proportion.
7. The method according to claim 4, characterized in that: In step d2), the primer F3, the primer R3 and the probe 3 in the primer set are mixed together in a molar ratio of 2:2:
1.
8. A method for identifying or assisting in identifying whether a test bacterium is a sphaeroides sporothrix, comprising the following steps: 1) extracting nucleic acid from the bacteria to be tested, using the nucleic acid from the bacteria to be tested as a template, and performing real-time fluorescence quantitative PCR using the primer set of claim 1; 2) After the reaction is complete, determine whether the test bacteria is Coccidioides by using the amplification curve and CT value: If the test bacteria has an S-shaped amplification curve and the Ct value is less than or equal to 37, the test bacteria is or is a candidate for Coccidioides; if the Ct value is greater than 40, the test bacteria is not or is not a candidate for Coccidioides; samples with Ct values between 37 and 40 require repeated testing; if the Ct value is still less than 40 in the repeated test, it is determined to be Coccidioides; if the Ct value exceeds 40, the test bacteria is not or is not a candidate for Coccidioides.
9. A method for identifying or assisting in identifying whether a sample contains Sporothrix sphaeroides, comprising the following steps: 1) extracting nucleic acid from a sample to be tested, and performing real-time fluorescence quantitative PCR using the nucleic acid as a template and the primer set according to claim 1; 2) After the reaction is complete, determine whether the test bacteria is sphaerotheca by using the amplification curve and CT value: If the sample to be tested has an S-shaped amplification curve and the Ct value is less than or equal to 37, the sample to be tested contains coccidioides; if the Ct value is greater than 40, the sample to be tested does not contain coccidioides; samples with Ct values between 37 and 40 need to be tested again; if the Ct value is still lower than 40 in the repeated test, the sample to be tested contains coccidioides, and if the Ct value exceeds 40, the sample to be tested does not contain coccidioides.
10. The method according to claim 8 or 9, characterized in that The real-time fluorescence quantitative PCR reaction is carried out in a PCR system, and the contents of the reaction system and the primer-probe combination in the PCR system meet the following conditions: 1) The PCR reaction system consisted of: 12.5 μL of qPCR premix, 1 μL each of primer F3 and primer R3 (final concentration 10 μM), 0.4 μL of probe 3 (final concentration 10 μM), 2 μL of template DNA, and RNase-free ddH2O to 25 μL. 2) The PCR reaction conditions were as follows: 1 cycle of pre-denaturation at 95°C for 10 min, followed by 50 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 15 s; the fluorescence signal was collected during the extension phase of each cycle.