Primer group and method for detecting malassezia furfur

Abstract

The invention relates to the technical field of biology, in particular to a primer group and a method for detecting malassezia furfur. The primer group for detecting malassezia furfur, disclosed by the invention, comprises the nucleotide sequences of primers as shown in SEQ ID NO. 1 to SEQ ID NO. 5; a skin sample can be directly collected and added into an LAMP reaction reagent integrated in the micro-fluidic chip, the detection process is automatically completed by a full-automatic detection instrument, a spectrum is analyzed through a spectrum sensor, comprehensive judgment is conducted on collected spectrum signals through an AI technology, full-automatic operation is achieved, the detection time can be controlled within one hour, and the detection efficiency is greatly improved. The kit realizes instant diagnosis, does not need professionals and laboratory environments, is portable and light, can be applied to instant detection in scenes such as pet hospitals on site, adopts an innovative LAMP test method, does not need nucleic acid extraction, and realizes sample outcome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer set and a method for detecting Malassezia furfur. Background Art

[0002] Malassezia furfur (M. furfur) infections typically present as localized skin diseases in immunocompetent individuals. The most common form is tinea versicolor (also known as tinea versicolor), characterized by circular or irregular hypopigmented or hyperpigmented macules on the trunk, neck, or upper limbs, accompanied by fine scaling and occasional mild pruritus. Alternatively, the organism may cause seborrheic dermatitis, characterized by erythema, greasy scaling, and pruritus on the scalp, face (such as the nose and between the eyebrows), or chest and back. These symptoms are generally mild and can be effectively controlled with topical antifungal medications (such as ketoconazole lotion). The prognosis is good, and systemic infection is rare.

[0003] However, the pathogenicity of Malassezia furfur is significantly enhanced in immunocompromised individuals (such as AIDS patients, those who use immunosuppressants or glucocorticoids for a long time), newborns (especially premature infants), and patients in intensive care units. Such people may develop deep or disseminated infections, such as fungemia (more common in newborns receiving intravenous fat emulsion therapy or patients with central venous catheters) or systemic infections. In addition, the bacteria can cause "neonatal capitis" in newborns, which manifests as sterile pustules on the head and face, which can be easily misdiagnosed as bacterial infections and require fungal microscopy or culture for definitive diagnosis. For people with severe immunodeficiency (such as hematopoietic stem cell transplant recipients or patients with advanced tumors), Malassezia furfur infection may progress to drug-resistant skin lesions or recurrent systemic infections. Therefore, high-risk populations need to undergo fungal testing (such as microscopy, PCR) as soon as possible when suspected symptoms appear to guide precise treatment.

[0004] To date, the most common and convenient method for detecting the presence of M. furfur is direct microscopy. This involves collecting a sample of skin scrapings or lesions, dissolving the stratum corneum with potassium hydroxide (KOH), and then directly observing the bacterial morphology under a microscope. This method is simple to use and produces rapid results, but it relies on operator experience and may result in false negatives, necessitating further testing to confirm the diagnosis. Furthermore, this method has certain limitations, as it requires operator proficiency during sampling and sample handling, as well as exposure to hazardous chemicals. The microscope required for observation further limits the convenience of on-site testing.

[0005] Testers can also use the molecular diagnostic technique of polymerase chain reaction (PCR) to diagnose M. furfur in samples. This method is currently a relatively effective method with a high detection rate for M. furfur. Using primers targeting conserved regions of the M. furfur genome or designing primers based on the M. furfur 28S rRNA sequence, and then using PCR to detect M. furfur, the results are highly specific and sensitive. PCR technology is widely used due to its high sensitivity, wide range of applicability, and ease of operation. However, the complex procedure, the need for sophisticated instrumentation, and the lengthy testing time make it unsuitable for on-site testing in non-laboratory settings and for widespread application in grassroots laboratories.

[0006] At present, patent publication number CN108148834A discloses primers and kits that can be used for loop-mediated isothermal amplification, and the primer group finally selected by the LAMP detection kit according to NCBI BLAST result is amplified sequence with Malassezia 28S rRNA (Sequence ID: KU729140.1) 11-243, a total of 233 bases, which can detect 34 kinds of fungi simultaneously. However, it cannot achieve the accurate diagnosis of Malassezia furfur. In addition, although the LAMP detection kit simplifies the nucleic acid amplification step by isothermal amplification, it is necessary to detect the LAMP reaction result by fluorescence or agarose gel electrophoresis detection, and the sample purity is relatively high, so it is still necessary to first perform nucleic acid extraction, then perform nucleic acid amplification, and the operating process is relatively complicated and cumbersome, and professional laboratory personnel are still needed to operate, and it is impossible to be directly used by pet doctors and nurses. Summary of the Invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a primer set and method for detecting Malassezia furfur.

[0008] In one aspect, the present invention provides a primer set for detecting Malassezia furfur, wherein the nucleotide sequence of the primers included in the primer set is as follows:

[0009] SEQ ID NO.1: 5'-CCTGGCTTTTGCTTGGTGTA-3';

[0010] SEQ ID NO.2: 5'-GCCACTTAAAGCCATTACGC-3';

[0011] SEQ ID NO.3:

[0012] 5'-AGGGGCCACATTCCGACTATTTTTTGGGTAGCAAGTCAGCATTG-3';

[0013] SEQ ID NO.4:

[0014] 5'-ATCTAGACCAAGGAACGCAGCGTTTTATCCTAAGCGCGAAGGTGT-3';

[0015] SEQ ID NO. 5: 5'-GCTTCTCCGACGATCCAAAC-3'.

[0016] Among them, the forward outer primer F3, its nucleotide sequence is shown in SEQ ID NO.1; the reverse outer primer B3, its nucleotide sequence is shown in SEQ ID NO.2; the forward inner primer FIP, its nucleotide sequence is shown in SEQ ID NO.3; the reverse inner primer BIP, its nucleotide sequence is shown in SEQ ID NO.4, and the loop primer LF, its nucleotide sequence is shown in SEQ ID NO.5.

[0017] Specifically, the present invention also provides the application of the above-mentioned primer set, which can be used to prepare a Malassezia furfur kit, or the application of the primer set in detecting Malassezia furfur, the application of the above-mentioned primer set in a microfluidic chip for detecting Malassezia furfur, and especially the detection of Malassezia furfur using a loop-mediated isothermal amplification-spectral sensor-artificial intelligence technology platform.

[0018] On the other hand, the present invention also provides a method for detecting Malassezia furfur, comprising: performing loop-mediated isothermal amplification on the sample to be tested using LAMP detection technology in combination with the primer set described above; and analyzing the amplified product.

[0019] Furthermore, the reaction temperature of the loop-mediated isothermal amplification is 65° C., and the reaction time is 30 minutes.

[0020] Furthermore, the loop-mediated isothermal amplification reaction is carried out in a microfluidic chip.

[0021] Furthermore, the method for analyzing the amplification product includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction results.

[0022] The beneficial effect of the present invention is that, for the 28S rRNA gene sequence of Malassezia furfur, the present invention has innovatively developed a set of specific primer formulas that can directly use fecal samples for LAMP reaction without nucleic acid extraction, and collect data through a spectral sensor to perform a method for rapid nucleic acid detection of Malassezia furfur. This method can directly use skin samples and add them to the LAMP reaction reagent integrated in the microfluidic chip. The detection process is automatically completed by a fully automatic detection instrument, which analyzes the spectrum through a spectral sensor and uses AI technology to make a comprehensive judgment on the collected spectral signals to achieve fully automatic operation, control the detection time to within one hour, and achieve instant diagnosis. It does not require professionals and laboratory environments, is portable and lightweight, and can be applied on-site for immediate detection in scenarios such as pet hospitals. It uses an innovative LAMP test method, does not require nucleic acid extraction, and can achieve sample-in, result-out.

[0023] Compared to the prior art, the present invention targets the nucleic acid sequence corresponding to the 28S rRNA of Malassezia furfur. The primer set finally selected is based on the 28S rRNA of Malassezia furfur with a total of 205 bases from 417 to 621. It is different from the target sequence of the existing LAMP detection kit and has high specificity for Malassezia furfur. In addition, a set of specific primer formulas that can directly use skin samples for LAMP reaction without nucleic acid extraction has been innovatively developed. The method collects data through a spectral sensor to perform a rapid nucleic acid detection method for M. fur. Compared with the existing LAMP kit detection method, the method is easy to operate. It only requires adding a sample to the test chip to fully automatically detect M. fur in the skin sample. It is simple to operate and only requires simple training for the detection personnel to achieve fully automatic detection of sample input and result output. It is suitable for rapid and instant detection in scenarios such as pet hospitals.

[0024] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention are realized and obtained by the structures particularly pointed out in the description and the drawings.

[0025] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is the screening result of LAMP reaction using 5 primer sets designed based on the 18S rRNA gene sequence of Malassezia furfur;

[0028] Figure 2 It is the test result of the present invention for CRY positive samples and negative samples;

[0029] Figure 3 is the detection result of the specific experiment of the present invention;

[0030] Figure 4 It is the detection result of the Malassezia furfur detection method used for skin samples of the present invention. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] In the embodiments of the present invention, the M.fur standard positive samples are synthetic M.fur-ITS gene sequence positive plasmids and M.fur-28S rRNA gene sequence positive plasmids, Salmonella (Sal) and Escherichia coli (EHEC) are both synthetic plasmid samples, and the primer sequences involved are all ordered from Sangon Biotech (Shanghai) Co., Ltd.

[0033] 1. Primer design and screening

[0034] Using a high concentration of M. fur standard positive samples, the following LAMP primer sets designed using M. fur-ITS nucleic acid fragments and M. fur-28S rRNA as templates were screened. The goal was to identify a primer set with optimal sensitivity and specificity.

[0035] The primer design method is:

[0036] (1) Download the 28S rRNA gene sequence (Sequence ID: KU729140.1) and ITS gene sequence (Sequence ID: NR_149347.1) of Malassezia furfur from NCBI as templates for primer design.

[0037] The 28S rRNA nucleic acid sequence (SEQ ID NO.6) is:

[0038]

[0039] The ITS nucleic acid sequence (SEQ ID NO.7) is:

[0040]

[0041]

[0042] (2) Using PrimerExplorer V5

[0043] Specific primers were designed using the 28S rRNA gene and ITS gene sequences of Malassezia furfur as templates (http: / / primerexplorer.jp / lampv5e / index.html). LAMP primers were designed based on six distinct regions of the 28S rRNA gene and ITS gene sequences, identified as F3, F2, F1, B1, B2, and B3, starting from the 5' end. F1, F2, F3, B1, B2, and B3 are approximately 20-bp long fragments of the target gene, and F1c and B1c are complementary to F1 and B1, respectively. The four primers include the upstream outer primer F3 (Forward Outer Primer), the downstream outer primer B3 (Backward Outer Primer), the upstream inner primer FIP (Forward Inner Primer), and the downstream inner primer BIP (Backward Inner Primer). At the same time, use the free software PrimerExplorer V5 to design 1-2 loop primers LF (Loop Primer Forward) and LB (Loop Primer Backward) between F1c and F2c, or between B1c and B2c to speed up the LAMP reaction. The primers are required to meet the T m The melting temperature of DNA chain is 64℃~66℃, and the T m The value is 59℃~61℃, loop primer T mThe free energy of the 3' end of F2 / B2, F3 / B3, and LF / LB, and the 5' end of F1c and B1c, should be no greater than -4 kcal / mol. The change in free energy (ΔG) is equal to the product free energy after primer-template binding minus the initial reactant free energy. The smaller the ΔG (the larger the absolute value of the negative number), the easier it is for the primer to bind to the template. The GC content of the primer sequence should be controlled between 45% and 65%. Also, ensure that each primer, especially the inner primer, is not prone to forming secondary structures to prevent the formation of primer-dimers. The sequence fragments recognized by each primer should have a certain appropriate spacing. The spacing between the F2 end and the B2 end (LAMP amplification region) should be 120 to 160 bases, the spacing between the 5' end of F2 and the 5' end of F1 (the region forming the loop structure) should be 40 to 60 bases, and the spacing between F2 and F3 should be 0 to 60 bases. All of the above conditions can be automatically calculated by the free software PrimerExplorer V5, and a list of designed primers will be provided.

[0044] (3) Multiple primer sets designed by PrimerExplorer V5 using the 28S rRNA gene sequence of Malassezia furfur and different positions on the ITS gene as templates were selected, and the target product sequences of different primer sets were input into NCBINucleotide BLAST

[0045] The online tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=Bl astSearch&LINK_LOC=blasthome) was used to analyze the specificity of the primer set corresponding to the LAMP product to ensure that there was no nonspecific amplification with other pathogens.

[0046] Through the above method, 7 sets of LAMP primer sets were preliminarily designed for selection. The nucleotide sequences of each primer set are shown in Table 1 below:

[0047] Table 1 Primer design

[0048]

[0049]

[0050] The above 7 primer sets were used to perform LAMP amplification experiments using M. fur standard positive samples as positive samples and sterile deionized water (ddH2O) as negative samples, and the color changes of the LAMP reaction system of each primer set were observed. Figure 1As shown in the figure, the primer set of group 3 changed the color of the positive sample (+) in a relatively short period of time (purple-red to bright yellow) and the negative sample (-) did not change color after 40 minutes, indicating that this primer set has higher sensitivity and specificity, while the other primer sets showed confusing positive and negative reactions or slow reaction speeds and were therefore not selected; in summary, group 3 is the primer set with the best sensitivity and specificity, therefore, group 3 primer set was selected as the primer set for detecting Malassezia furfur.

[0051] 2. Reaction conditions

[0052] After several rounds of testing under different reaction conditions, the following conditions were finally selected as the final reagent ratio and reaction conditions:

[0053] (1) Preparation of 10× primer mixture:

[0054] Table 2 Primer set mixing concentration ratio

[0055] Working concentration (μM) 10× primer mix concentration (μM) FIP 1.6 16 BIP 1.6 16 F3 0.2 2 B3 0.2 2 LF 0.4 4

[0056] Table 3

[0057] Primer concentration (μM) Primer name Prepare 40 reactions (μL) 100 FIP 8 100 BIP 8 10 F3 10 10 B3 10 100 LF 2 Sterile deionized water 12 Total volume (μL) 50

[0058] (2) Primers, 2×LAMP premix, and positive / negative sample ratios in the LAMP reaction system:

[0059] Table 4

[0060]

[0061]

[0062] The 2×LAMP premix solution has a well-known formula, including MgSO 4 (MgSO 4 concentration is 6 mM to 12 mM), buffer, dNTPs, KCl, (NH 4 ) 2 SO 4 , Bst DNA polymerase, phenol red, sterile enzyme-free water, etc.

[0063] The positive sample can be a M.fur standard positive sample, that is, a synthetic M.fur-28S rRNA gene sequence positive plasmid.

[0064] The negative sample can be sterile deionized water (ddH2O).

[0065] (4) Reaction conditions

[0066] Under the condition of 65℃ temperature, constant temperature amplification for 30 minutes can achieve better detection results.

[0067] Heating can be performed using a constant temperature heating device (water bath, metal bath, constant temperature oven, or PCR instrument, etc.).

[0068] 3. LAMP detection method for Malassezia furfur (M.fur)

[0069] The present invention uses a M.fur standard positive sample and performs detection according to the following steps:

[0070] Step 1: M.fur positive / negative sample preparation

[0071] The concentration of the M. fur standard positive sample was diluted to 500 copies / μL as a positive sample; sterile deionized water (ddH2O) was used as a negative sample.

[0072] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0073] Step 3: Take the Malassezia furfur (M. fur) positive / negative samples prepared in the first step and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0074] Step 4: Heat at 65°C and react for 30 minutes.

[0075] The reaction results are as follows Figure 2 As shown, the LAMP reaction systems of the 20 negative samples (bottom) were all purple-red, and the LAMP reaction systems of the 20 positive samples (top) were all yellow. The positive accuracy rate and the negative accuracy rate in the reaction results were 100%, indicating that Malassezia furfur can be clearly detected.

[0076] 4. Specificity Experiment

[0077] In order to detect the specificity of the detection primer set for Malassezia furfur, the primers were used at high concentrations (10 7 CFU / mL) of Salmonella (Sal) and Escherichia coli (EHEC) plasmid solutions were used as cross-detection samples.

[0078] Step 1: Preparation of M.fur positive / negative samples for cross-testing

[0079] Positive sample: A M. fur standard positive sample diluted to a concentration of 500 copies / μL was used as a positive sample;

[0080] Negative samples: diluted to a concentration of 10 7 CFU / mL of Salmonella (Sal) and Escherichia coli (EHEC) plasmid solutions were used as negative samples.

[0081] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0082] Step 3: Take the positive samples and negative samples prepared in the first step respectively, and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0083] Step 4: Heat at 65°C and react for 30 minutes.

[0084] The reaction results are as follows Figure 3 As shown, the LAMP reaction systems of the four positive samples containing the M. fur standard positive samples all turned yellow, while the five negative samples containing the Salmonella (Sal) plasmid solution (upper left) and the five negative samples containing the Escherichia coli (EHEC) plasmid solution (lower left) did not change color. It can be seen that no false positive results occurred, demonstrating that the primer set for detecting Malassezia furfur of the present invention has good specificity.

[0085] according to Figure 2 and Figure 3 The sensitivity and specificity of LAMP for detecting Malassezia furfur were preliminarily calculated based on the experimental results. Figure 2 and Figure 3 According to the experimental results, 24 out of 24 positive samples showed clear positive results, so the sensitivity was calculated as: As well as 30 of the negative samples of 10 other bacterial species and 20 sterile deionized water (ddH2O), all showed clear negative results, so the specificity was calculated as:

[0086] 5. LAMP detection method for Malassezia furfur (M.fur) in skin samples

[0087] Step 1: Preparation of positive / negative skin samples

[0088] Use a cotton swab to wipe different parts of the skin 3 to 5 times. Immerse the cotton swab containing the skin sample completely in 2.5 mL of pre-prepared sterile deionized water. Shake the cotton swab dozens of times to dissolve the skin sample in the water to obtain a negative skin sample.

[0089] A certain dilution ratio of M. fur standard positive sample (concentration of 10 5 CFU / mL) was added to a portion of the negative skin sample prepared above to obtain a positive skin sample.

[0090] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0091] Step 3: The positive / negative skin samples prepared in the first step were used to prepare the LAMP reaction systems for each reaction according to the method in Table 4 above.

[0092] Step 4: Heat at 65°C and react for 30 minutes.

[0093] The reaction results are as follows Figure 4 As shown, all 10 negative skin samples (-) showed clear negative results, and all 10 positive skin samples (+) turned yellow, indicating that Malassezia furfur in the skin samples could be clearly detected.

[0094] The LAMP detection method for Malassezia furfur in skin samples is simple in collecting and processing skin samples, without the need for nucleic acid extraction. The sample processing method is simple, without the need for liquid nitrogen grinding, ultrasonic fragmentation and other nucleic acid extraction methods, which simplifies the detection steps. At the same time, the addition of loop primers increases the isothermal amplification reaction rate and shortens the detection time. It only takes about 1 hour from sample collection to test results.

[0095] This detection method can also detect Malassezia furfur through the loop-mediated isothermal amplification-spectral sensor-artificial intelligence (LAMP-SpectralSensor-AI) technology platform. The diluted sample to be tested can be directly injected into the LAMP reaction reagent integrated in the microfluidic chip reaction chamber for amplification. The amplified product is analyzed by the spectral sensor, and the collected spectral signals are comprehensively judged by AI technology to quickly determine whether Malassezia furfur is present in the sample. The spectral detection method is combined with the AI ​​algorithm to make the detection accuracy reach the level of molecular detection. By limiting the sample to be tested to the microfluidic environment of the device, the risk of sample contamination is reduced, and the sample volume and reagents required for detection are minimized, thereby further reducing the overall cost of screening and testing. Moreover, this technology does not require any complex operations and can be completed on-site by medical staff in pet hospitals with simple training. It can realize fully automatic detection of sample input and result output, which is suitable for rapid and instant detection in scenarios such as pet hospitals.

[0096] With the above-described preferred embodiments of the present invention as a guide, and with reference to the above description, relevant personnel are fully capable of making various changes and modifications without departing from the technical scope of this invention. The technical scope of this invention is not limited to the contents of the specification and must be determined according to the scope of the claims.

Claims

1. A primer set for detecting Malassezia furfur, characterized in that, The nucleotide sequences of the primers included in the primer set are as follows: SEQ ID NO.1: 5'-CCTGGCTTTTGCTTGGTGTA-3'; SEQ ID NO.2: 5'-GCCACTTAAAGCCATTACGC-3'; SEQ ID NO.3: 5'-AGGGGCCACATTCCGACTATTTTTTGGGTAGCAAGTCAGCATTG-3'; SEQ ID NO.4: 5'-ATCTAGACCAAGGAACGCAGCGTTTTATCCTAAGCGCGAAGGTGT-3'; SEQ ID NO. 5: 5'-GCTTCTCCGACGATCCAAAC-3'.

2. Application of the primer set according to claim 1 in preparing a Malassezia furfur detection kit.

3. Application of the primer set according to claim 1 in detecting Malassezia furfur.

4. Application of the primer set according to claim 1 in a microfluidic chip for detecting Malassezia furfur.

5. A method for detecting Malassezia furfur, characterized in that, include: Performing loop-mediated isothermal amplification on the sample to be tested using LAMP detection technology combined with the primer set as claimed in claim 1; Analyze the results of amplification products.

6. The detection method of Malassezia furfur according to claim 5, wherein The reaction temperature of the loop-mediated isothermal amplification is 65° C., and the reaction time is 30 minutes.

7. The method for detecting Malassezia furfur according to claim 5, wherein The loop-mediated isothermal amplification reaction is carried out in a microfluidic chip.

8. The method for detecting Malassezia furfur according to claim 5, wherein The method for analyzing the results of the amplification products includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction results.

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