A kit for detecting microorganisms and use thereof

Abstract

The present application relates to a kit for microbial detection and application thereof. The present application relates to a detection primer probe combination, which comprises a first primer pair for detecting bacteria and a first probe, the first primer pair comprises a forward primer shown as SEQ ID NO: 52 or 53 and a reverse primer shown as SEQ ID NO: 54 or 55, and the first probe has a probe shown as SEQ ID NO: 56. The kit provided by the present application has the advantages of high sensitivity, high specificity, rapidness and high efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene detection, in particular to a kit for microorganism detection and application thereof. BACKGROUND

[0002] Traditional microorganism contamination detection mainly relies on culture method, biochemical identification and microscope observation. Culture method (such as agar plate culture and broth enrichment) is the gold standard, but the detection period is long (1-3 days for bacteria, 3-7 days for fungi, and even 21 days for mycoplasma), and it cannot detect unculturable or slowly growing microorganisms. Biochemical identification (such as API strip and mass spectrometry) can improve accuracy, but it still relies on culture and has limited ability to distinguish mixed contamination. Microscope examination (such as Gram staining and fluorescent staining) is fast but has low sensitivity and is prone to miss low-level contamination. In addition, mycoplasma detection usually uses culture method combined with DNA fluorescent staining (such as Hoechst 33258), but the operation is complicated and time-consuming. These methods are difficult to meet the needs of rapid and high-sensitivity detection in biological product production.

[0003] With the continuous development of medical microbiology research technology, pathogenic diagnosis is no longer limited to the level of pathogen, and detection methods at the molecular and gene levels are constantly emerging and being applied in clinical and laboratory. Although the existing molecular detection methods and kits for microorganisms meet the clinical needs to some extent, there are still some problems and shortcomings. The existing commercial kits generally only detect a certain specific microorganism, have limited detection range, high cost and long time consumption, and cannot detect bacteria, fungi and mycoplasma at the same time; at the same time, for samples with low microorganism content, false negatives are prone to occur, and the sensitivity is extremely low, especially for the detection of fungi and viruses, some species cannot be detected, which affects the accuracy of the final detection result. Moreover, although the existing PCR technology shortens the detection time, it still has problems such as high false positive rate and insufficient sensitivity due to unreasonable primer probe design.

[0004] Therefore, there is an urgent need in the art for a detection method with high sensitivity, high specificity and rapidity, which can accurately identify microorganisms. SUMMARY

[0005] In view of the defects involved in the prior art, the present application provides a method and kit for detecting bacteria, fungi and mycoplasma in one PCR system, which is rapid, has high sensitivity, wide species coverage and can simultaneously detect bacteria, fungi and mycoplasma.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] A first object of the present application is to provide a primer probe combination (or primer probe composition) for microbial detection, the primer probe combination comprising a first primer pair and a first probe for detecting bacteria, the first primer pair comprising a forward primer as shown in SEQ ID NO: 52 or 53 and a reverse primer as shown in SEQ ID NO: 54 or 55, and the first probe having a probe as shown in SEQ ID NO: 56.

[0008] Preferably, the primer probe combination further comprises a second primer pair and a second probe for detecting fungi, the second primer pair comprising a forward primer as shown in SEQ ID NO: 13 or 14 and a reverse primer as shown in SEQ ID NO: 15 or 16, and the second probe having a probe as shown in SEQ ID NO: 17 or 18.

[0009] Preferably, the primer probe combination further comprises a third primer pair and a third probe for detecting mycoplasma, the third primer pair comprising a forward primer as shown in SEQ ID NO: 28 or 29 and a reverse primer as shown in any one of SEQ ID NO: 30 to 32, and the third probe having a probe as shown in SEQ ID NO: 33.

[0010] Preferably, the primer probe combination further comprises a fourth primer pair and a fourth probe for detecting an internal reference gene, the fourth primer pair comprising a forward primer as shown in SEQ ID NO: 4 and a reverse primer as shown in SEQ ID NO: 5, and the fourth probe having a probe as shown in SEQ ID NO: 6.

[0011] Preferably, the first primer pair comprises a forward primer as shown in SEQ ID NO: 52 and a reverse primer as shown in SEQ ID NO: 55.

[0012] Preferably, the second primer pair comprises a forward primer as shown in SEQ ID NO: 13 and a reverse primer as shown in SEQ ID NO: 15, and the second probe has a probe as shown in SEQ ID NO: 18.

[0013] Preferably, the second primer pair comprises a forward primer as shown in SEQ ID NO: 14 and a reverse primer as shown in SEQ ID NO: 16, and the second probe has a probe as shown in SEQ ID NO: 18.

[0014] Preferably, the third primer pair comprises a forward primer as shown in SEQ ID NO: 29 and a reverse primer as shown in SEQ ID NO: 30.

[0015] A second object of the present application is to provide a kit comprising the primer probe combination according to any one of the above.

[0016] A third object of the present application is to provide the use of the primer probe combination according to any one of the above or the kit according to the above in microbial detection.

[0017] Preferably, the microorganism comprises one or more of bacteria, fungi, mycoplasma.

[0018] Preferably, the bacteria comprises one or more of Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, Bacillus megaterium.

[0019] Preferably, the fungi comprises one or more of Saccharomyces cerevisiae, Aspergillus niger, Candida albicans, Aspergillus brasiliensis, Candida glabrata, Candida tropicalis, Aspergillus flavus, Aspergillus oryzae, Candida kefyr, Kluyveromyces lactis, Tremella fuciformis, Aspergillus ruber, Malassezia furfur, Lentinula edodes, Trichophyton mentagrophytes.

[0020] Preferably, the mycoplasma comprises Mycoplasma orale and / or Mycoplasma pneumoniae.

[0021] A fourth object of the present application is to provide a method for detecting microorganisms, comprising the following steps:

[0022] The sample to be tested is subjected to a nucleic acid amplification reaction using the primer probe combination according to any one of the above or the kit according to the above, thereby obtaining a detection result.

[0023] Effects of the application:

[0024] The primer probe combination provided by the present application can simultaneously detect bacteria, fungi and mycoplasma, has significant detection specificity, and has higher detection sensitivity, with a detection limit of 1-5 CFU / mL for bacteria, fungi and mycoplasma, and is suitable for trace contamination detection with relaxed requirements for the type and quality of the sample; has wide coverage and can detect a variety of microorganisms (such as Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, Bacillus megaterium, Saccharomyces cerevisiae, Aspergillus niger, Candida albicans, Aspergillus brasiliensis, Candida glabrata, Candida tropicalis, Aspergillus flavus, Aspergillus oryzae, Candida kefyr, Kluyveromyces lactis, Tremella fuciformis, Aspergillus ruber, Malassezia furfur, Lentinula edodes, Trichophyton mentagrophytes, Mycoplasma orale and Mycoplasma pneumoniae).

[0025] The primer probe combination provided by the present application can simultaneously detect bacteria, fungi and mycoplasma in one PCR system, greatly shortening the detection period (completed within 2 hours), reducing the consumption of samples and the operation steps, and has great industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 This is a multi-component amplification pattern of Bacillus subtilis in Example 5;

[0027] Figure 2 This is a multi-component amplification map of Lactobacillus casei in Example 5;

[0028] Figure 3 This is a multi-component amplification map of Mycoplasma pneumoniae in Example 5;

[0029] Figure 4 This is a multi-component amplification map of oral mycoplasma in Example 5;

[0030] Figure 5 This is a multi-component amplification pattern of sample DNA-1 from Example 6;

[0031] Figure 6 This is a multi-component amplification pattern of DNA-2 from sample 6;

[0032] Figure 7 This is a multi-component amplification pattern of DNA-3 from sample 7 in Example 7;

[0033] Figure 8 This is a multi-component amplification pattern of DNA-4 from sample 7 in Example 7;

[0034] Figure 9 This is a multi-component amplification pattern of bacteria detected by the commercially available kit in Example 8;

[0035] Figure 10 This is the amplification multicomponent pattern of the sample (bacteria) detected by the kit of the present invention in Example 8;

[0036] Figure 11 This is a multi-component amplification spectrum of fungi detected by the commercially available kit in Example 8;

[0037] Figure 12 This is the amplification multicomponent spectrum of the sample (fungus) detected by the kit of the present invention in Example 8;

[0038] Figure 13 The image shows the amplified multicomponent spectrum of mycoplasma detected by the commercially available kit in Example 8;

[0039] Figure 14 This is the amplification multicomponent spectrum of the sample (mycoplasma) detected by the kit of the present invention in Example 8. Detailed Implementation

[0040] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0041] Addressing the issues of high false positive rates and insufficient sensitivity due to inadequate primer and probe design in existing qPCR methods, the inventors, through extensive and in-depth research, screening, and experimentation, particularly through primer and probe optimization, and combined with a quantitative real-time PCR (qPCR) platform, have developed a rapid, efficient, highly sensitive, and specific primer-probe combination and kit for simultaneously detecting bacteria, fungi, and mycoplasma in a single PCR system. The primers and probes provided in this invention are designed based on qPCR technology. During the detection process, an internal reference gene (such as human GAPDH) is incorporated to monitor the efficiency of the entire process and avoid false negatives. The probes are labeled with different fluorescent groups (such as FAM and HEX) to achieve multi-target detection in a single tube. It is suitable for screening microbial contamination in biopharmaceuticals such as pharmaceuticals, vaccines, and cell therapy products, exhibiting high sensitivity, high specificity, and rapid detection. Based on this, this invention was completed.

[0042] The first object of the present invention is to provide a primer-probe combination (or primer-probe composition) for microbial detection, the primer-probe combination comprising a first primer pair and a first probe for detecting bacteria, the first primer pair comprising a forward primer shown in SEQ ID NO: 52 or 53 and a reverse primer shown in SEQ ID NO: 54 or 55, and the first probe having a probe shown in SEQ ID NO: 56.

[0043] In some embodiments, the primer-probe combination further includes a second primer pair and a second probe for detecting fungi, the second primer pair including the forward primer shown in SEQ ID NO: 13 or 14 and the reverse primer shown in SEQ ID NO: 15 or 16, and the second probe having the probe shown in SEQ ID NO: 17 or 18.

[0044] In some embodiments, the primer-probe combination further includes a third primer pair and a third probe for detecting mycoplasma, the third primer pair including the forward primer shown in SEQ ID NO: 28 or 29 and the reverse primer shown in any of SEQ ID NO: 30 to 32, and the third probe having the probe shown in SEQ ID NO: 33.

[0045] In some embodiments, the bacteria include one or more of Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, and Bacillus megaterium.

[0046] In some embodiments, the fungi include one or more of the following: Saccharomyces cerevisiae, Aspergillus niger, Candida albicans, Aspergillus brasiliensis, Candida glabrata, Candida tropicalis, Aspergillus flavus, Aspergillus oryzae, Candida krusei, Kluyveromyces lactis, Tremella fuciformis, Aspergillus rubrum, Malassezia furfur, Lentinus shiitake, and Trichophyton mentagrophytes.

[0047] In some embodiments, the mycoplasma includes oral mycoplasma and / or pneumonia mycoplasma.

[0048] In some embodiments, the primer-probe combination further includes a fourth primer pair and a fourth probe for detecting an internal reference gene, the fourth primer pair including the forward primer shown in SEQ ID NO: 4 and the reverse primer shown in SEQ ID NO: 5, and the fourth probe having the probe shown in SEQ ID NO: 6.

[0049] In this invention, the fourth primer pair and the fourth probe used to detect the internal reference gene are designed for a specific conserved region of the human GAPDH gene.

[0050] In this invention, the primers and probes for each microorganism listed in Table 1 can be used in combination. For example, for bacteria, there are two forward primers (SEQ ID NO: 52 or 53) and two reverse primers (SEQ ID NO: 54 or 55). The first primer pair used to detect bacteria includes: the reverse primer composed of the forward primer shown in SEQ ID NO: 52 and SEQ ID NO: 54, the reverse primer composed of the forward primer shown in SEQ ID NO: 52 and SEQ ID NO: 55, the reverse primer composed of the forward primer shown in SEQ ID NO: 53 and SEQ ID NO: 54, and the reverse primer composed of the forward primer shown in SEQ ID NO: 53 and SEQ ID NO: 55.

[0051] In some embodiments, the first primer pair includes the forward primer shown in SEQ ID NO: 52 and the reverse primer shown in SEQ ID NO: 55.

[0052] In some embodiments, the first primer pair includes the forward primer shown in SEQ ID NO: 53 and the reverse primer shown in SEQ ID NO: 55.

[0053] In some embodiments, the first primer pair includes the forward primer shown in SEQ ID NO: 52 and the reverse primer shown in SEQ ID NO: 54.

[0054] In some embodiments, the first primer pair includes the forward primer shown in SEQ ID NO: 53 and the reverse primer shown in SEQ ID NO: 54.

[0055] In some embodiments, the second primer pair includes the forward primer shown in SEQ ID NO: 13 and the reverse primer shown in SEQ ID NO: 15, and the second probe has the probe shown in SEQ ID NO: 18.

[0056] In some embodiments, the second primer pair includes the forward primer shown in SEQ ID NO: 13 and the reverse primer shown in SEQ ID NO: 16, and the second probe has the probe shown in SEQ ID NO: 18.

[0057] In some embodiments, the second primer pair includes the forward primer shown in SEQ ID NO: 14 and the reverse primer shown in SEQ ID NO: 15, and the second probe has the probe shown in SEQ ID NO: 18.

[0058] In some embodiments, the second primer pair includes the forward primer shown in SEQ ID NO: 14 and the reverse primer shown in SEQ ID NO: 16, and the second probe has the probe shown in SEQ ID NO: 18.

[0059] In some embodiments, the second primer pair includes the forward primer shown in SEQ ID NO: 13 and the reverse primer shown in SEQ ID NO: 15, and the second probe has the probe shown in SEQ ID NO: 17.

[0060] In some embodiments, the third primer pair includes the forward primer shown in SEQ ID NO:29 and the reverse primer shown in SEQ ID NO:30.

[0061] In some embodiments, the third primer pair includes the forward primer shown in SEQ ID NO:29 and the reverse primer shown in SEQ ID NO:31.

[0062] In some embodiments, the third primer pair includes the forward primer shown in SEQ ID NO:29 and the reverse primer shown in SEQ ID NO:32.

[0063] In some embodiments, the third primer pair includes the forward primer shown in SEQ ID NO:28 and the reverse primer shown in SEQ ID NO:30.

[0064] In some embodiments, the third primer pair includes the forward primer shown in SEQ ID NO:28 and the reverse primer shown in SEQ ID NO:31.

[0065] In some embodiments, the third primer pair includes the forward primer shown in SEQ ID NO:28 and the reverse primer shown in SEQ ID NO:32.

[0066] In this invention, the primer or probe sequence contains degenerate bases (i.e., designed to contain two or more different bases at the same site), for example, Y represents C / T, R represents A / G, etc.

[0067] In some embodiments, the 5' and 3' ends of the first, second, third, and fourth probes respectively and independently contain a fluorescent group and a quenching group.

[0068] In a preferred embodiment, the probe has a fluorescent group attached to its 5' end and a quenching group at its 3' end.

[0069] In this invention, each probe in the primer-probe set uses a different fluorescent group, which does not affect the detection of each other. That is, different channels can be used for detection. For example, FAM, JOE, ROX, HEX, CY5 and Q705 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected so that they will not interfere with each other.

[0070] The primer-probe combination described above is designed based on the qPCR platform.

[0071] In some embodiments, the components of the primer-probe combination are each contained in a separate package.

[0072] In some embodiments, the components of the primer-probe combination are contained in the same package.

[0073] In some embodiments, the components of the primer-probe combination exist in a mixed form.

[0074] A second object of the present invention is to provide a kit comprising the primer-probe combination described in any of the preceding claims.

[0075] In some embodiments, the kit also includes negative and positive controls.

[0076] A third object of the present invention is to provide the use of the primer-probe combination or the kit described above in microbial detection.

[0077] In some embodiments, the microorganisms include one or more of bacteria, fungi, and mycoplasma.

[0078] In some embodiments, the bacteria include one or more of Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, and Bacillus megaterium.

[0079] In some embodiments, the fungi include one or more of the following: Saccharomyces cerevisiae, Aspergillus niger, Candida albicans, Aspergillus brasiliensis, Candida glabrata, Candida tropicalis, Aspergillus flavus, Aspergillus oryzae, Candida krusei, Kluyveromyces lactis, Tremella fuciformis, Aspergillus rubrum, Malassezia furfur, Lentinus shiitake, and Trichophyton mentagrophytes.

[0080] In some embodiments, the mycoplasma includes oral mycoplasma and / or pneumonia mycoplasma.

[0081] A fourth objective of this invention is to provide a method for detecting microorganisms, the method comprising the following steps:

[0082] Using the primer-probe combination as described above or the kit as described above, a nucleic acid amplification reaction is performed on the sample to be tested to obtain the detection result.

[0083] In this invention, the presence of bacteria, fungi, and mycoplasma contamination in the sample is determined based on the shape of the amplification curve and the CT value. The judgment criteria are as follows: if the amplification curve is a typical S-shape and the CT value is ≤35, the sample is positive; if the CT value is >35 or not detected, the sample is negative.

[0084] In some embodiments, the method further includes DNA extraction from the sample to be tested. In this invention, there are no particular limitations on the method of DNA extraction; any method well-known in the art, such as the kit method, can be used. After obtaining the DNA, it is preferable to perform quality control on the DNA, including testing for concentration and integrity. The DNA that passes the tests is diluted and refrigerated for later use. Using the extracted DNA as a template, this invention utilizes the primers and probes to prepare a qPCR reaction system for qPCR amplification.

[0085] In this invention, the nucleic acid amplification reaction system (qPCR) contains hot-start Taq enzyme, dNTPs, and Mg. 2+ Buffer. The qPCR system of this invention is prepared as follows: 10 μL of 2×Perfect Probe qPCR Master Mix (probe-based qPCR amplification premix), primer probe (2 μL), ROX reference dye (2 μM, 2 μL), template DNA (1 μL), and water is added to bring the total to 20 μL.

[0086] In some embodiments, the concentration of the first primer pair is 5 to 30 μM, for example 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, or 30 μM.

[0087] In some embodiments, the concentration of the second primer pair is 5 to 40 μM, for example 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, or 40 μM.

[0088] In some embodiments, the concentration of the third primer pair is 5 to 40 μM, for example 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, or 40 μM.

[0089] In some implementations, the concentration ratio of the first primer pair to the third primer pair in the qPCR system is 1 to 2:1, such as 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0090] In a preferred embodiment, the second primer pair and the third primer pair have the same concentration.

[0091] In some embodiments, the concentration of the first probe is 5 to 20 μM, for example, 5 μM, 10 μM, 15 μM, or 20 μM.

[0092] In some embodiments, the concentration of the second probe is 5 to 30 μM, for example 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, or 30 μM.

[0093] In some embodiments, the concentration of the third probe is 5 to 15 μM, for example, 5 μM, 10 μM, or 15 μM.

[0094] In some embodiments, the concentration ratio of the first probe to the second probe is 1:1 to 2, such as 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0095] In some embodiments, the concentration ratio of the first probe to the second probe is 1:1 to 1.5.

[0096] In some embodiments, the concentration ratio of the first probe to the third probe is 1:0.1 to 2, such as 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0097] In this invention, the reaction program for nucleic acid amplification is 95°C for 5 min; 95°C for 15 s; 60°C for 1 min; 35 cycles.

[0098] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0099] Example 1 Primer and probe design

[0100] This embodiment designs specific primers and probes based on the conserved gene sequences of common contaminating microorganisms (such as bacteria, fungi, and mycoplasma) in bioproducts, and screens and tests the primers and probes (see Table 1). During detection, a fluorescent reporter gene is attached to the 5' end of the probe sequence, and a fluorescence quencher group is attached to the 3' end.

[0101] Primer and probe design for the internal control: Primers and probes were screened from multiple human genes to ensure no interaction with the microbial primers and probes used for detection and to achieve optimal amplification efficiency. The resulting internal control sequences are shown in Table 1 and are used as the internal control sequences in the kit. The upstream and downstream primers and probes used to detect the internal control were designed targeting specific conserved regions of the human GAPDH gene.

[0102] In this invention, the sequence information of the primer-probe combination is shown in Table 1:

[0103] Table 1

[0104]

[0105]

[0106] In this embodiment, the primers and probes listed in Table 1 were used to detect human (10 ng / μL), bacterial (1 ng / μL), fungal (1 ng / μL), and mycoplasma DNA (0.1 ng / μL), respectively. DNA of different concentrations was added to the reaction solution, and a blank control group (NC group, water) was also established.

[0107] Preparation of PCR amplification system (Table 2):

[0108] Table 2

[0109]

[0110] The reaction procedure for real-time quantitative PCR is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 60℃ for 1 min, for 40 cycles.

[0111] The detection results of different primers and different samples are shown in Table 3. The primer amplification effect was judged based on the CT value and specificity. Among them, the bacteria in Table 3 are Bacillus subtilis (serial number 8) or Bacillus megaterium (serial numbers 9-11), the fungus is Aspergillus niger, and the mycoplasma is Mycoplasma stomatitis.

[0112] Table 3

[0113]

[0114] The results showed that the detection effect of fungi was good when the primers and probes of the present invention were F-F1-2, F-R1-1, F-P1-1 and F-F1, F-R1-2, F-P1-1; the detection effect of mycoplasma was good when the primers and probes of the present invention were M-F2-1, M-R2-1, M-P2; and the detection effect of bacteria was good when the primers and probes were B-F5-1, B-R5-1, B-5P, B-F5-2, B-R5-1, B-5P and B-F5-2, B-R5-2, B-5P.

[0115] Example 2 Validation of universal bacterial primers

[0116] This embodiment uses the primers and probes described above, which exhibit excellent detection performance, to detect bacterial DNA:

[0117] DNA was extracted from three bacteria—Bacillus subtilis, Lactobacillus casei, and Lactobacillus plantarum—using a nucleic acid extraction kit (Takekin EE401-01). The extracted bacterial DNA was then quantitatively analyzed. The extracted bacterial DNA was serially diluted to concentrations of 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL. Different concentrations of bacterial DNA were added to the reaction solution, and a blank control group (NC group, water) was also established.

[0118] Preparation of PCR amplification system (Table 4):

[0119] Table 4

[0120]

[0121] The reaction procedure for real-time quantitative PCR is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 60℃ for 1 min, for 35 cycles.

[0122] The test results for different concentrations and different samples are shown in Table 5. Result interpretation: if the CT value is ≤35, it indicates that the sample is positive; if the CT value is >35 or not detected, it indicates that the sample is negative.

[0123] Table 5

[0124]

[0125] The results showed that the primers and probes designed in this invention have excellent detection effects and good amplification effect on low concentrations of DNA. In particular, when the concentration of the detected microorganisms is extremely low (0.1 pg / μL), the primers and probes designed in this application can still detect Bacillus subtilis, Lactobacillus casei, and Lactobacillus plantarum, which proves the feasibility of the kit and method of this invention and that it has higher sensitivity.

[0126] Example 3: Validation of universal primers for fungi and mycoplasma

[0127] DNA was extracted from *Saccharomyces cerevisiae*, *Aspergillus niger*, *Mycoplasma pneumoniae*, and *Mycoplasma oralis* using the same method as in Example 2, and the DNA was serially diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL. Different concentrations of fungal or mycoplasma DNA were added to the reaction solution, while a blank control group (NC group, water) was established.

[0128] In this embodiment, the primers and probes used for fungal and mycoplasma detection are F-F1-2, F-R1-1, and F-P1-1 (fungi), and M-F2-1, M-R2-1, and M-P2 (mycoplasma) as shown in Table 1. The PCR amplification system is shown in Table 4. The only difference is the concentration of the probes and primers. In this embodiment, primers and probes F-F1-2, F-R1-1, and F-P1-1 are all 8 μM; M-F2-1, M-R2-1, and M-P2 are all 8 μM.

[0129] The amplification procedure is as in Example 2. The fungal detection results are shown in Table 6, and the mycoplasma detection results are shown in Table 7. The result interpretation method is the same as in Example 2.

[0130] Table 6

[0131]

[0132] Table 7

[0133]

[0134] The results showed that the primers and probes designed in this invention have excellent detection effects on fungi and mycoplasma, and also have good amplification effects on low concentrations of DNA. In particular, when the concentration of the detected microorganisms is extremely low (0.1 pg / μL), the primers and probes designed in this application can detect Saccharomyces cerevisiae, Aspergillus niger, Mycoplasma pneumoniae, and Mycoplasma stomatitis, which proves the feasibility of the kit and method of this invention, as well as its higher sensitivity and reduced false negatives.

[0135] Example 4 Detection of different fungi

[0136] DNA was extracted from *Saccharomyces cerevisiae*, *Aspergillus niger*, *Candida albicans*, *Aspergillus brasiliensis*, *Candida glabrata*, *Candida tropicalis*, *Aspergillus flavus*, *Aspergillus oryzae*, *Candida krusei*, *Kluyveromyces lactis*, *Tremella fuciformis*, *Aspergillus rubrum*, *Malassezia furfur*, *Lentinula edodes*, and *Trichophyton mentagrophytes* according to the method in Example 2, and diluted to 1 ng / μL.

[0137] Following the fungal PCR amplification system in Example 3, the amplification procedure was the same as in Example 2, the result interpretation method was the same as in Example 2, and the detection results are shown in Table 8:

[0138] Table 8

[0139]

[0140] As shown in Table 8, the primers, probes and kits provided by this invention amplified the DNA of 15 fungi well, indicating that the primers in the kit of this invention can correctly detect common fungi.

[0141] Example 5: Differentiation between bacterial and mycoplasma contamination

[0142] Bacterial detection primers: B-F5-1, B-R5-2, B-5P; mycoplasma detection primers: M-F2-1, M-R2-1, M-P2. Simultaneously, a fluorescent dye (FAM) was attached to the 5' end of the bacterial probe (B-5P), and a fluorescent dye (JOE) was attached to the 5' end of the mycoplasma probe (M-P2). The two sets of primers and probes were mixed to form a 10× primer mix. Bacterial DNA (Bacillus subtilis and Lactobacillus casei) and mycoplasma DNA (Mycoplasma pneumoniae and Mycoplasma stomatitis) were extracted according to the method in Example 2. The amplification system was prepared according to Table 9, where the concentration of each microbial DNA was 1 ng / μL.

[0143] Table 9

[0144]

[0145] The amplification procedure and result interpretation method are as described in Example 2. The results are shown in Table 10. Multi-component amplification maps of Bacillus subtilis, Lactobacillus casei, Mycoplasma pneumoniae, and Mycoplasma oralis are shown below.Figures 1 to 4 .

[0146] Table 10

[0147]

[0148] In this embodiment, when the sample contains bacteria, bacterial DNA can be well amplified, while mycoplasma primers do not produce a peak; when the sample contains mycoplasma, mycoplasma DNA can be well amplified, while bacterial primers do not produce a peak, indicating that the primer-probe-kit composite amplification primers provided by the present invention have good specificity and can effectively amplify the target genes.

[0149] Example 6: Detection of Microbial Contamination in Cell Culture Medium

[0150] Select cell culture medium (purchased from Zhuhai Beso Cell Science Co., Ltd.) and Mycoplasma pneumoniae. Add 20 μL of Mycoplasma pneumoniae bacterial solution (10...) to 380 μL of cell culture medium. 6 Mix 400 μL of uncontaminated cell culture medium (DNA-1) with CFU / mL. Simultaneously, set up a 400 μL uncontaminated control group (DNA-2). Extract DNA using a nucleic acid extraction kit (Takes EE401-01 from TransGen), following the instructions. Take 1 ng / μL each of the above Bacillus subtilis DNA, Aspergillus niger DNA, and Mycoplasma pneumoniae DNA and mix them as a positive reference (PC). Add water to the blank control to create the neutral (NC).

[0151] Primers for bacterial detection were selected as follows: B-F5-1, B-R5-2, and B-5P; primers for fungal detection were selected as follows: F-F1-2, F-R1-1, and F-P1-1; and primers for mycoplasma detection were selected as follows: M-F2-1, M-R2-1, and M-P2. Simultaneously, fluorescent dyes (FAM) were attached to the 5' end of the bacterial probe (B-5P), fluorescent dyes (JOE) were attached to the 5' end of the mycoplasma probe (M-P2), and fluorescent dyes (ROX) were attached to the 5' end of the fungal probe (F-P1-1). The three sets of primers and probes were mixed to form a 10× primer mix. The amplification system was prepared according to Table 11, where the DNA concentration was 1 ng / μL.

[0152] Table 11

[0153]

[0154] The amplification procedure and result interpretation method are as described in Example 2. The results are shown in Table 12. The multi-component amplification patterns of DNA-1 and DNA-2 are shown in Table 12. Figures 5 to 6 .

[0155] Table 12

[0156]

[0157] In this embodiment, when mycoplasma contamination is present in the cell culture medium, the primers, probes, or kits provided by this invention can effectively amplify its DNA, indicating that this invention can detect mycoplasma in the sample and proving the feasibility of the method provided by this invention.

[0158] Example 7: Detection of Microbial Contamination in Cosmetics

[0159] Select ginseng exosomes (extracted in-house) and Aspergillus niger from the laboratory. Take 380 μL of ginseng exosomes and add 20 μL of Aspergillus niger (10... 7 Mix CFU / ml (DNA-3), and simultaneously set up a 400 μL uncontaminated control group of ginseng exosomes (DNA-4). DNA was extracted according to the method in Example 6, and an NC group and a positive control group (PC) were set up in accordance with Example 6.

[0160] Referring to the amplification system in Table 11 and the amplification procedure in Example 2, the microorganisms in the test sample were detected. The result interpretation method was the same as in Example 2, and the detection results are shown in Table 13. The multi-component amplification patterns of DNA-3 and DNA-4 are shown in Table 13. Figures 7 to 8 .

[0161] Table 13

[0162]

[0163] In this embodiment, when cosmetics containing exosomes are contaminated with fungi, the primers, probes, or kits provided by this invention can effectively amplify their DNA, indicating that this invention can detect fungi in the sample and proving the feasibility of the method provided by this invention.

[0164] Example 8: Comparison of detection results with commercially available kits

[0165] The primer-probe combination provided by this invention was compared with the bacterial DNA detection kit (ZY002P007) and fungal DNA detection kit (ZY002P006) from Nanjing Zhengyang, and the mycoplasma DNA detection kit (A.1.001-02) from Pucai Biotechnology. Commercially available kits were operated according to their instructions.

[0166] 1 ng / μL of Bacillus subtilis DNA, Saccharomyces cerevisiae DNA, and Mycoplasma pneumoniae DNA were added to the reaction system. The primers and PCR amplification system used in this example are shown in Table 11, the amplification procedure is as in Example 2, the result interpretation method is as in Example 2, and the results are shown in Table 14. The multi-component amplification patterns of each kit are shown in Table 14. Figures 9 to 14 .

[0167] Table 14

[0168]

[0169] The results above show that the biggest advantage of the primer-probe combination or kit provided by this invention compared to the three commercially available kits is that a single PCR system can simultaneously detect bacterial, fungal, and mycoplasma contamination, making it more convenient and efficient. Meanwhile, the Zhengyang bacterial DNA detection kit and fungal detection kit showed poor amplification, and the negative control showed a peak, which not only hindered interpretation but also potentially led to misjudgments and affected the accuracy of the test results. Using the kit of this invention, negative samples showed no amplification, effectively avoiding false positive results. Compared to the kit from Pucai Biotechnology, the mycoplasma DNA detection kit had a larger Ct value and a less smooth curve, indicating that this kit is more sensitive.

[0170] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A primer probe combination for microbial detection, characterized by, the primer probe combination comprises a first primer pair and a first probe for detecting bacteria, a second primer pair and a second probe for detecting fungi, a third primer pair and a third probe for detecting mycoplasma; the first probe is a probe as shown in SEQ ID NO: 56, and the first primer pair comprises a forward primer as shown in SEQ ID NO: 52 and a reverse primer as shown in SEQ ID NO: 55; the second probe is a probe as shown in SEQ ID NO: 18, and the second primer pair comprises a forward primer as shown in SEQ ID NO: 13 and a reverse primer as shown in SEQ ID NO: 15; the third probe is a probe as shown in SEQ ID NO: 33, and the third primer pair comprises a forward primer as shown in SEQ ID NO: 29 and a reverse primer as shown in SEQ ID NO:

30.

2. The primer probe combination according to claim 1, characterized in that, the primer probe combination further comprises a fourth primer pair and a fourth probe for detecting an internal reference gene, the fourth primer pair comprises a forward primer as shown in SEQ ID NO: 4 and a reverse primer as shown in SEQ ID NO: 5, and the fourth probe has a probe as shown in SEQ ID NO:

6.

3. A kit characterized in that, the kit comprises the primer probe combination of any one of claims 1-2.

4. Use of the primer probe combination of any one of claims 1-2 or the kit of claim 3 in the detection of microorganisms for non-diagnostic and non-therapeutic purposes; the microorganism is one or more of bacteria, fungi, and mycoplasma; the bacteria is one or more of Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, Bacillus megaterium; the fungi is one or more of Saccharomyces cerevisiae, Aspergillus niger, Candida albicans, Aspergillus brasiliensis, Candida glabrata, Candida tropicalis, Aspergillus flavus, Aspergillus oryzae, Candida krusei, Kluyveromyces lactis, Tremella fuciformis, Aspergillus ruber, Malassezia furfur, Lentinula edodes, Trichophyton mentagrophytes; the mycoplasma is Mycoplasma orale and / or Mycoplasma pneumoniae.

5. A method of detecting microorganisms for non-diagnostic, non-therapeutic purposes, characterized in that the method comprises the following steps: performing a nucleic acid amplification reaction on the sample to be tested using the primer probe combination of any one of claims 1-2 or the kit of claim 3, thereby obtaining a detection result; the microorganism is one or more of bacteria, fungi, and mycoplasma; the bacteria is one or more of Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, Bacillus megaterium; the fungi is one or more of Saccharomyces cerevisiae, Aspergillus niger, Candida albicans, Aspergillus brasiliensis, Candida glabrata, Candida tropicalis, Aspergillus flavus, Aspergillus oryzae, Candida krusei, Kluyveromyces lactis, Tremella fuciformis, Aspergillus ruber, Malassezia furfur, Lentinula edodes, Trichophyton mentagrophytes; the mycoplasma is Mycoplasma orale and / or Mycoplasma pneumoniae.

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