A microbial detection kit and detection method

Abstract

This invention discloses a microbial detection kit and detection method, belonging to the field of microbial detection technology. The kit includes a microbial nucleic acid extraction solution, a PCR reaction solution, a positive control, and a negative control. The extraction solution contains synergistic components such as guanidine isothiocyanate and betaine, and the PCR reaction solution contains four target microorganism-specific primer pairs. The detection method includes sample pretreatment, nucleic acid extraction, PCR amplification, and result interpretation. This invention achieves simultaneous detection of four microorganisms, including Escherichia coli, through the synergistic effect of its components. The detection sensitivity reaches 1 CFU / mL, with 100% specificity and a detection cycle of 70-90 minutes. The kit exhibits excellent stability, is easy to operate and prepare, and is suitable for various scenarios such as food, medical, and environmental applications. It addresses the pain points of existing technologies, such as single detection, low sensitivity, and long cycle times, and has significant industrial value.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a microbial detection kit and detection method. Background Technology

[0002] Microbial contamination is a key factor affecting food safety, drinking water hygiene, the accuracy of clinical diagnosis and treatment, and environmental quality. Rapid and accurate detection of microbial species and concentrations is a core means of preventing microbial transmission and controlling contamination risks. Currently, conventional methods for microbial detection mainly include traditional culture methods, biochemical identification methods, and ordinary nucleic acid amplification methods. These methods have many limitations in practical applications and cannot meet the needs for efficient and accurate detection.

[0003] Traditional culture methods, as classic approaches to microbial detection, rely on the growth characteristics of microorganisms for isolation, culture, and identification. However, they suffer from a fatal flaw: long detection cycles. They cannot provide rapid early warning of contamination, making them particularly unsuitable for time-sensitive scenarios such as emergency clinics and rapid food release. Furthermore, these methods are cumbersome, requiring multiple manual steps including inoculation, culture, and biochemical reactions, resulting in high labor costs. They also have extremely low detection rates for unculturable or slow-growing microorganisms, leading to frequent false negatives. Biochemical identification methods differentiate microorganisms by detecting their metabolites. While this can shorten detection time to some extent, it still relies on the initial culture process and has poor specificity, easily affected by impurities in the sample. It struggles to accurately distinguish between microorganisms with similar morphology and metabolites, resulting in a high false positive rate. In addition, these methods have low throughput, detecting only one or a few microorganisms per test, failing to meet the need for simultaneous detection of multiple pathogens and creating a significant mismatch with the high-efficiency detection requirements of clinical and industrial applications. While existing nucleic acid amplification test kits have addressed the long detection cycles of traditional methods, they still suffer from insufficient sensitivity, weak anti-interference capabilities, and complex preparation processes. Most kits use conventional primer and probe designs, which have limited detection capabilities for low concentrations of microorganisms. Furthermore, sample pretreatment is cumbersome, requiring the addition of lysis reagents, and the components within the kits have poor stability, easily becoming ineffective at room temperature, necessitating refrigerated transport and storage, increasing detection costs and operational complexity. In addition, existing technologies are mostly designed for single bacterial species, lacking the ability to specifically identify target microorganisms in complex samples, and are susceptible to interference from other microorganisms.

[0004] To address the aforementioned problems, invention patent document CN119846208B discloses a microbial detection kit and detection method. The kit includes an ELISA plate coated with a capture antibody and an HRP-labeled antibody working solution. The capture antibody is a monoclonal antibody LG3A-1 that specifically binds to human Giardia lamblia antigen, with its heavy chain amino acid sequence shown in SEQ ID NO. 1 and its light chain amino acid sequence shown in SEQ ID NO. 2. The HRP-labeled antibody is a monoclonal antibody FLSP4 that specifically binds to human Giardia lamblia antigen, with its heavy chain amino acid sequence shown in SEQ ID NO. 3 and its light chain amino acid sequence shown in SEQ ID NO. 4. The microbial detection kit provided by this invention can detect the content of Giardia lamblia in human feces, exhibiting high specificity, high sensitivity, good stability, and simple operation, and has excellent application prospects. However, its detection range is limited, only able to detect Giardia lamblia in human feces, and it cannot achieve simultaneous detection of multiple common pathogenic microorganisms. Furthermore, it does not address the industry pain points of missed detection of low-concentration microorganisms and low detection efficiency, making it difficult to adapt to the detection needs of various scenarios such as food, drinking water, and environmental samples.

[0005] Therefore, developing a microbial detection kit and detection method that combines high sensitivity, short cycle time, and strong specificity has significant practical and industrial value. Summary of the Invention

[0006] In view of the above, the present invention aims to provide a microbial detection kit and detection method. The kit has a simple preparation process, stable components, strong anti-interference ability, and a rapid, accurate, and convenient detection method, which can realize the simultaneous detection of multiple microorganisms.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a microbial detection kit, comprising microbial nucleic acid extraction solution, PCR reaction solution, positive control, negative control, stop solution and detection buffer; Preferably, each 100 mL of the microbial nucleic acid extract consists of the following raw materials: 15-20 g guanidine isothiocyanate, 18-22 mL 1 mol / L Tris-HCl buffer (pH=8.0), 0.5-0.8 g EDTA-Na2, 1-1.5 mL Triton X-100, 98-102 μL RNase A (10 mg / mL), 0.3-0.5 g betaine, 0.05-0.1 g L-cysteine, 0.1-0.15 g ascorbic acid, with the remainder being enzyme-free deionized water; Preferably, each 25 μL of the PCR reaction solution consists of the following raw materials: 12-13 μL of 2×Taq PCR MasterMix, 0.4-0.6 μL each of 10 μmol / L Escherichia coli-specific primer pairs, 0.4-0.6 μL each of 10 μmol / L Staphylococcus aureus-specific primer pairs, 0.4-0.6 μL each of 10 μmol / L Salmonella-specific primer pairs, 0.4-0.6 μL each of 10 μmol / L Candida albicans-specific primer pairs, 0.2-0.4 μL of SYBR Green I fluorescent dye, and the remainder is enzyme-free deionized water; Preferably, the upstream primer sequence of the *E. coli*-specific primer pair is 5'-GGTGATGCTGGTGCTGATAA-3', and the downstream primer sequence is 5'-CTGCTGGTGGTGGTGGTGAT-3'; the upstream primer sequence of the *Staphylococcus aureus*-specific primer pair is 5'-AGAAGCGTTATCCGGAATGA-3', and the downstream primer sequence is 5'-CAGCGGTTTGTCCTTGTAAC-3'; the upstream primer sequence of the *Salmonella*-specific primer pair is 5'-ATGAAGAAAGCGAAAGCGGA-3', and the downstream primer sequence is 5'-TCGCTGCGTTCTTCATCAGT-3'; and the upstream primer sequence of the *Candida albicans*-specific primer pair is 5'-GAGAGTTTGATCCTGGCTCAG-3', and the downstream primer sequence is 5'-TGCACCATCTGTCACTCTGTTA-3'. Preferably, the positive control is a standard nucleic acid solution of Escherichia coli, Staphylococcus aureus, Salmonella, and Candida albicans, with a concentration of 100 ng / μL. The Escherichia coli strain is Escherichia coli K-12 MG1655, the Staphylococcus aureus strain is Staphylococcus aureus ATCC 25923, the Salmonella strain is Salmonella typhimurium ATCC14028, and the Candida albicans strain is Candida albicans ATCC 90028. Preferably, the negative control is enzyme-free deionized water; the stop solution is a 0.4-0.6 mol / L EDTA-Na2 solution with pH=8.0; and the detection buffer consists of the following ingredients per 100 mL: 25-35 mL of 1 mol / L Tris-HCl buffer with pH=7.5, 5-6 g of NaCl, 0.25-0.35 g of MgCl2, with the remainder being enzyme-free deionized water.

[0008] Preferably, each 100 mL of the microbial nucleic acid extract further includes the following raw materials: Tween-20 0.1-0.2 mL, bovine serum albumin 0.2-0.4 g, polyethylene glycol PEG 6000 0.1-0.3 g, NP-40 0.08-0.15 mL, and D-sorbitol 0.2-0.3 g.

[0009] Another object of the present invention is to provide a detection method using the above-mentioned microbial detection kit, comprising the following steps: Step S1, Sample Pretreatment: Food, drinking water, medical, and environmental samples are pretreated accordingly to obtain ready-to-use sample solutions; Step S2, Microbial nucleic acid extraction: Take 1 mL of the prepared sample solution, add 0.8-1.2 mL of microbial nucleic acid extraction solution, place at room temperature for 9-12 min, centrifuge at 11000-13000 r / min for 10-12 min, aspirate the supernatant and add 1 mL of detection buffer, mix well, and set aside. Step S3, PCR amplification: Take a reaction tube containing 20-24 μL of PCR reaction solution, add 2-4 μL of nucleic acid solution, positive control, and negative control respectively, mix well and centrifuge, and then put it into a real-time fluorescence quantitative PCR instrument for PCR amplification; Step S4, Result Judgment: The negative control has no amplification curve and a Ct value > 38; the positive control has an obvious amplification curve and a Ct value < 38; the dissolution curve is single and without impurities; the sample with a Ct value ≤ 38 and a dissolution curve consistent with the corresponding positive control is positive; otherwise, it is negative. After the test, stop solution is added to terminate the reaction.

[0010] Preferably, the PCR amplification program in step S3 is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, for a total of 40 cycles; melting curve analysis at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s.

[0011] Preferably, the pretreatment in step S1 is as follows: When the sample is food, take 25g of sample and add 225mL of 0.9% sterile saline, homogenize for 2min to obtain a 1:10 homogenate, serially dilute and take 1mL for later use; When the sample is drinking water, take 10mL of sample, centrifuge at 8000r / min for 10min, discard the supernatant, and resuspend the precipitate with 1mL of sterile saline for later use; When the sample is medical sputum, add an equal volume of sterile saline, shake to mix, centrifuge at 8000r / min for 10min, discard the supernatant and resuspend the precipitate; For medical blood samples: take 1mL, add 3mL of erythrocyte lysis buffer, shake for 10min, centrifuge at 8000r / min for 10min, discard the supernatant and resuspend the precipitate; When the sample is environmental soil, take 10g and add 90mL of sterile saline, shake for 30min, let stand for 10min, and take 1mL of supernatant for later use; When the sample is environmental sewage, take 10mL of... Centrifuge at 8000 r / min for 10 min, discard the supernatant and resuspend the precipitate for later use.

[0012] Due to the application of the above technical solution, the present invention has the following beneficial effects: (1) The microbial detection kit and detection method disclosed in this invention overcome many limitations of the prior art through the scientific ratio and synergistic effect of each component, and achieve positive technical gains. Compared with traditional detection methods and existing nucleic acid amplification kits, it has achieved significant improvements in detection efficiency, sensitivity, specificity and adaptability. Among them, the raw materials in the microbial nucleic acid extract are not simply superimposed, but form a synergistic technical system: guanidine isothiocyanate, as the core lysis component, can efficiently destroy the microbial cell wall structure. Betaine works synergistically with it, which can not only further enhance the lysis effect, but also effectively protect the nucleic acid from denaturation and avoid detection failure caused by nucleic acid degradation. L-cysteine ​​and ascorbic acid form an antioxidant synergistic system, which can remove reactive oxygen free radicals generated during sample lysis, reduce nucleic acid chain damage, and at the same time help improve the cell wall lysis efficiency. Combined with guanidine isothiocyanate and betaine, the purity and recovery rate of nucleic acid extraction are significantly improved, which solves the technical pain points of low purity and insufficient recovery rate of nucleic acid extraction in existing kits.

[0013] (2) The microbial detection kit and detection method disclosed in this invention achieve simultaneous and accurate detection of four common pathogenic microorganisms by optimizing the component ratio of the PCR reaction solution and primer design. This breaks through the limitations of single-species detection in existing technologies and significantly improves detection sensitivity and specificity, effectively solving the industry problems of missed detection of low-concentration microorganisms and false positives caused by interference from other microorganisms. Among them, the four specific primer pairs show no cross-hybridization after sequence alignment, which can specifically amplify the corresponding microbial nucleic acid. Combined with the precise tracing effect of SYBR Green I fluorescent dye and synergistic with 2×Taq PCRMasterMix, the amplification time is further shortened, making the entire detection process shorter. Compared with the traditional culture method (24-72h) and existing nucleic acid detection kits (more than 120min), the detection efficiency is greatly improved, making it suitable for time-sensitive scenarios such as emergency clinical care and rapid food release.

[0014] (3) The microbial detection kit and detection method disclosed in this invention, with the addition of Tween-20, bovine serum albumin, polyethylene glycol PEG 6000, NP-40, D-sorbitol and other components in the microbial nucleic acid extraction solution, form a multi-dimensional synergistic effect with the original components, further optimizing the overall performance of the kit: Tween-20 and Triton X-100 synergistically enhance the decontamination ability and reduce the adsorption of nucleic acids by impurities in the sample; bovine serum albumin can bind to the protease in the extraction solution, inhibit the degradation of nucleic acids by the protease, and stabilize the activity of RNase A, avoiding enzyme inactivation affecting the removal effect of RNA impurities; polyethylene glycol PEG6000 can promote nucleic acid precipitation and further improve the nucleic acid recovery rate; D-sorbitol maintains the osmotic pressure stability of the extraction system and protects the nucleic acids from being damaged during the lysis of microbial cells. The synergistic effect of these components significantly improves the integrity and stability of nucleic acid extraction, while extending the shelf life of the kit, solving the problems of poor component stability, strict low-temperature storage and transportation, and high cost of existing kits.

[0015] (4) The microbial detection kit and detection method disclosed in this invention are easy to operate, requiring no complex pretreatment equipment. The sample pretreatment process has been optimized, and targeted pretreatment schemes have been designed for different types of samples such as food, drinking water, medical samples, and environmental samples. This ensures sufficient enrichment of target microorganisms in the samples, simplifies the operation steps, and reduces labor costs. Compared with the cumbersome sample pretreatment process in the prior art, it greatly improves the ease of operation and avoids detection errors caused by cumbersome operation. No professional high-end equipment is required, and it can be adapted to the detection needs of multiple scenarios such as food hygiene supervision, clinical testing, and environmental monitoring. It solves the problems of complex preparation and limited detection scenarios of existing kits, and has significant industrial value and application prospects. Detailed Implementation

[0016] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0017] Example 1: Preparation of Microbial Detection Kit 1.1 Instrument Preparation Instruments: Real-time quantitative PCR instrument (ABI 7500), high-speed centrifuge (Thermo Fisher), constant temperature shaker, homogenizer, sterile ultra-clean workbench, electronic balance (accuracy 0.001g), pH meter.

[0018] 1.2 Preparation of reagent kit components (1) Preparation of microbial nucleic acid extraction solution: Take enzyme-free deionized water to a final volume of 100 mL, add 18 g of guanidine isothiocyanate, and stir at 25 °C and 300 r / min until completely dissolved; add 20 mL of 1 mol / L Tris-HCl buffer (pH=8.0), stir for 10 min and adjust the pH to 8.0; add 0.6 g of EDTA-Na2, 1.2 mL of Triton X-100, 0.4 g of betaine, 0.08 g of L-cysteine, 0.12 g of ascorbic acid, 0.15 mL of Tween-20, 0.3 g of bovine serum albumin, 0.2 g of polyethylene glycol PEG 6000, 0.12 mL of NP-40, and 0.25 g of D-sorbitol, and stir for 20 min until completely dissolved; add 10 mg / mL of RNase A. 100 μL, gently invert and stir for 5 min; after sterilization by filtration through a 0.22 μm sterile filter membrane, dispense into 1 mL / tube and store at 4 °C.

[0019] (2) Preparation of PCR reaction solution: Under ice bath conditions, add 12.5 μL of 2×Taq PCR MasterMix, 0.5 μL each of the 10 μmol / L E. coli-specific primer pair (upstream 5'-GGTGATGCTGGTGCTGATAA-3', downstream 5'-CTGCTGGTGGTGGTGGTGAT-3'), and 0.5 μL each of the 10 μmol / L Staphylococcus aureus-specific primer pair (upstream 5'-AGAAGCGTTATCCGGAATGA-3', downstream 5'-CAGCGGTTTGTCCTTGTAAC-3') to each 25 μL system. Add 0.5 μL of 0.5 μL of Salmonella-specific primer pair (upstream 5'-ATGAAGAAAGCGAAAGCGGA-3', downstream 5'-TCGCTGCGTTCTTCATCAGT-3'), 0.5 μL of 10 μmol / L Candida albicans-specific primer pair (upstream 5'-GAGAGTTTGATCCTGGCTCAG-3', downstream 5'-TGCACCATCTGTCACTCTGTTA-3'), and 0.3 μL of SYBR Green I fluorescent dye. Add enzyme-free deionized water to a final volume of 25 μL, mix well, aliquot into 22 μL tubes, and store at -20°C.

[0020] (3) Preparation of positive control: Escherichia coli K-12 MG1655, Staphylococcus aureus ATCC25923, Salmonella typhimurium ATCC14028 and Candida albicans ATCC 90028 standard strains were cultured respectively. The nucleic acid of each strain was extracted using the microbial nucleic acid extraction solution prepared above. The concentration was adjusted to 100 ng / μL with enzyme-free deionized water, aliquoted into 100 μL / tube, and stored at -20℃.

[0021] (4) Preparation of negative control: Pack 100 μL of enzyme-free deionized water into tubes and store at 4°C.

[0022] (5) Preparation of the stop solution: Weigh 14.61g of EDTA-Na2, add enzyme-free deionized water to dissolve, adjust the pH to 8.0 with NaOH, make up to 100mL, the concentration is 0.5mol / L, filter to sterilize, dispense into 100μL / tube, and store at 4℃.

[0023] (6) Preparation of detection buffer: Take enzyme-free deionized water to make up to 100 mL, add 30 mL of 1 mol / L Tris-HCl buffer (pH=7.5), 5.85 g of NaCl, and 0.29 g of MgCl2, stir for 15 min until completely dissolved, adjust the pH to 7.5, filter to sterilize, dispense into 1 mL / tube, and store at 4℃.

[0024] 1.3 Kit Assembly: Place the prepared components (10 tubes of nucleic acid extraction solution, 20 tubes of PCR reaction solution, 4 tubes of positive control, 4 tubes of negative control, 10 tubes of stop solution, 10 tubes of detection buffer), instructions and sterile pipette tips into the kit, close the lid tightly and label, and store them separately in the refrigerator or freezer as required.

[0025] Example 2: Detection method and performance testing using the kit of the present invention. 2.1 Detection methods and procedures (taking food samples, drinking water samples, medical sputum samples, and environmental soil samples as examples) Step S1, Sample pretreatment: (1) Food sample (milk): Take 25g of milk sample, add 225mL of 0.9% sterile saline, homogenize for 2min to obtain a 1:10 homogenate, and serially dilute to 10. -3 Take 1 mL of the diluted solution as a spare sample solution.

[0026] (2) Drinking water sample: Take 10 mL of drinking water, centrifuge at 8000 r / min for 10 min, discard the supernatant, resuspend the precipitate with 1 mL of sterile physiological saline, and obtain the sample solution for use.

[0027] (3) Medical sputum sample: Take 1 mL of sputum sample, add 1 mL of sterile physiological saline, shake to mix, centrifuge at 8000 r / min for 10 min, discard the supernatant, resuspend the precipitate with 1 mL of sterile physiological saline, and obtain the sample solution for use.

[0028] (4) Environmental soil samples: Take 10g of soil sample, add 90mL of sterile physiological saline, shake in a constant temperature shaker for 30min, let stand for 10min, and take 1mL of supernatant as a spare sample solution.

[0029] Step S2, Microbial Nucleic Acid Extraction: Take 1 mL of each of the above-mentioned prepared sample solutions, add 1 mL of the microbial nucleic acid extraction solution prepared in Example 1, place at room temperature for 10 min, centrifuge at 12000 r / min for 11 min, aspirate 0.5 mL of the supernatant, add 1 mL of detection buffer and mix well to obtain the nucleic acid solution for later use.

[0030] Step S3, PCR amplification: Take a reaction tube containing 22 μL of PCR reaction solution, add 3 μL of the above nucleic acid solution, positive control, and negative control respectively, mix gently, and centrifuge at 10000 r / min for 30 s; place the reaction tube in a real-time fluorescence quantitative PCR instrument, and set the amplification program: pre-denaturation 95℃ 5 min; denaturation 95℃ 30 s, annealing 58℃ 30 s, extension 72℃ 30 s, for a total of 40 cycles; melting curve analysis 95℃ 15 s, 60℃ 1 min, 95℃ 15 s.

[0031] Step S4, Result Judgment: The negative control has no amplification curve and a Ct value > 38; the positive control has an obvious amplification curve and a Ct value < 38; the melting curve is single and without impurities; the sample Ct value ≤ 38 and the melting curve is consistent with the corresponding positive control is positive; otherwise, it is negative. After the test, add 5 μL of stop solution to each tube, mix gently, and terminate the reaction.

[0032] 2.2 Performance test data (3 parallel experiments, average value) (1) Sensitivity test: Standard bacterial solutions of Escherichia coli K-12 MG1655, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC14028 and Candida albicans ATCC 90028 were prepared and serially diluted to 1 CFU / mL, 5 CFU / mL, 10 CFU / mL and 100 CFU / mL. The above detection method was used for detection. Three replicate wells were set for each concentration. The test results are shown in Table 1 below.

[0033] As shown in Table 1, the detection sensitivity of the kit for the four microorganisms can reach 1 CFU / mL, and the positive detection rate is 100% at each concentration. The melting curve is single and without impurity peaks, indicating that the kit has high sensitivity and strong amplification specificity.

[0034] Table 1

[0035] (2) Specificity test: Ten common bacteria, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus subtilis, and Proteus, were selected and tested using the kit of this invention. Three replicate wells were set for each bacteria. Four positive and negative controls of the target microorganisms were also set. The test results are shown in Table 2 below.

[0036] As shown in Table 2, the kit of the present invention shows a positive reaction for only four target microorganisms and a negative reaction for 10 common bacteria, with a specificity of 100%. This effectively avoids false positives caused by interference from other bacteria and solves the problem of insufficient specificity in the prior art.

[0037] Table 2

[0038] (3) Stability test: The kit of the present invention was stored at 4℃ for 1 month, 3 months and 6 months, and frozen at -20℃ for 6 months and 12 months. At the same time, repeated freeze-thaw tests were carried out (frozen at -20℃ for 12 hours, thawed at room temperature for 2 hours, repeated 3 times) to test the detection performance of the kit. The freshly prepared kit was used as a control. The results are shown in Table 3 below.

[0039] Table 3

[0040] As shown in Table 3, the detection performance of the kit of the present invention did not decrease significantly after being refrigerated at 4℃ for 6 months and frozen at -20℃ for 12 months, and the positive detection rate was still 100%, with the coefficient of variation of Ct values ​​all being <4%. After repeated freeze-thaw cycles 3 times, it still maintained good detection performance, indicating that the kit components have excellent stability and solve the problems of poor stability and short shelf life of existing kits.

[0041] Example 3: Experiment to verify the synergistic effect of components To verify the synergistic effect of the components in the microbial nucleic acid extract, a control group (containing only the basic components: guanidine isothiocyanate, Tris-HCl buffer, EDTA-Na2, Triton X-100, RNase A) and an experimental group (containing the basic components plus newly added synergistic components: betaine, L-cysteine, ascorbic acid, Tween-20, bovine serum albumin, etc.) were set up. Escherichia coli nucleic acid was extracted from each group, and the purity of nucleic acid extraction (A260 / A280 ratio), recovery rate and nucleic acid integrity were tested. Each group was set up with 3 replicates. The results are shown in Table 4 below.

[0042] Table 4

[0043] As shown in Table 4, the nucleic acid extraction purity (A260 / A280 ratio close to the standard value of 1.8-2.0) and recovery rate of the experimental group (including the synergistic component) were significantly higher than those of the control group, and the nucleic acid integrity was better. This indicates that the newly added synergistic component in this invention has a good synergistic effect with the basic component, which can significantly improve the nucleic acid extraction effect. This effect is not a simple superposition of the individual effects of each component, but has a synergistic effect, which solves the technical pain point of poor nucleic acid extraction effect of existing kits.

[0044] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microbial detection kit, characterized in that, This includes microbial nucleic acid extract, PCR reaction solution, positive control, negative control, stop solution, and detection buffer; The microbial nucleic acid extract consists of the following ingredients per 100 mL: guanidine isothiocyanate 15-20 g, 18-22 mL of 1 mol / L Tris-HCl buffer (pH=8.0), EDTA-Na2 0.5-0.8 g, Triton X-100 1-1.5 mL, RNase A 10 mg / mL 98-102 μL, betaine 0.3-0.5 g, L-cysteine ​​0.05-0.1 g, ascorbic acid 0.1-0.15 g, with the remainder being enzyme-free deionized water. Each 25 μL of the PCR reaction solution consists of the following ingredients: 12-13 μL of 2×Taq PCR MasterMix, 0.4-0.6 μL each of 10 μmol / L Escherichia coli-specific primer pairs, 0.4-0.6 μL each of 10 μmol / L Staphylococcus aureus-specific primer pairs, 0.4-0.6 μL each of 10 μmol / L Salmonella-specific primer pairs, 0.4-0.6 μL each of 10 μmol / L Candida albicans-specific primer pairs, 0.2-0.4 μL of SYBR Green I fluorescent dye, and the remainder is enzyme-free deionized water; The upstream primer sequence of the *E. coli*-specific primer pair is 5'-GGTGATGCTGGTGCTGATAA-3', and the downstream primer sequence is 5'-CTGCTGGTGGTGGTGGTGAT-3'; the upstream primer sequence of the *Staphylococcus aureus*-specific primer pair is 5'-AGAAGCGTTATCCGGAATGA-3', and the downstream primer sequence is 5'-CAGCGGTTTGTCCTTGTAAC-3'; the upstream primer sequence of the *Salmonella*-specific primer pair is 5'-ATGAAGAAAGCGAAAGCGGA-3', and the downstream primer sequence is 5'-TCGCTGCGTTCTTCATCAGT-3'; the upstream primer sequence of the *Candida albicans*-specific primer pair is 5'-GAGAGTTTGATCCTGGCTCAG-3', and the downstream primer sequence is 5'-TGCACCATCTGTCACTCTGTTA-3'.

2. The microbial detection kit according to claim 1, characterized in that, The positive controls were standard nucleic acid solutions of Escherichia coli, Staphylococcus aureus, Salmonella, and Candida albicans, all at a concentration of 100 ng / μL. The Escherichia coli strain was Escherichia coli K-12 MG1655, the Staphylococcus aureus strain was Staphylococcus aureus ATCC 25923, the Salmonella strain was Salmonella typhimurium ATCC14028, and the Candida albicans strain was Candida albicans ATCC 90028.

3. The microbial detection kit according to claim 1, characterized in that, The negative control is enzyme-free deionized water; the stop solution is a 0.4-0.6 mol / L EDTA-Na2 solution with pH=8.0; the detection buffer consists of the following ingredients per 100 mL: 25-35 mL of 1 mol / L Tris-HCl buffer with pH=7.5, 5-6 g of NaCl, 0.25-0.35 g of MgCl2, and the remainder is enzyme-free deionized water.

4. The microbial detection kit according to claim 1, characterized in that, The microbial nucleic acid extract also includes the following ingredients per 100 mL: Tween-20 0.1-0.2 mL, bovine serum albumin 0.2-0.4 g, polyethylene glycol PEG 6000 0.1-0.3 g, NP-40 0.08-0.15 mL, and D-sorbitol 0.2-0.3 g.

5. A detection method using the microbial detection kit according to any one of claims 1-4, characterized in that, Includes the following steps: Step S1, Sample Pretreatment: Food, drinking water, medical, and environmental samples are pretreated accordingly to obtain ready-to-use sample solutions; Step S2, Microbial nucleic acid extraction: Take 1 mL of the prepared sample solution, add 0.8-1.2 mL of microbial nucleic acid extraction solution, place at room temperature for 9-12 min, centrifuge at 11000-13000 r / min for 10-12 min, aspirate the supernatant and add 1 mL of detection buffer, mix well, and set aside. Step S3, PCR amplification: Take a reaction tube containing 20-24 μL of PCR reaction solution, add 2-4 μL of nucleic acid solution, positive control, and negative control respectively, mix well and centrifuge, and then put it into a real-time fluorescence quantitative PCR instrument for PCR amplification; Step S4, Result Judgment: The negative control has no amplification curve and a Ct value > 38; the positive control has an obvious amplification curve and a Ct value < 38; the dissolution curve is single and without impurities; the sample with a Ct value ≤ 38 and a dissolution curve consistent with the corresponding positive control is positive; otherwise, it is negative. After the test, stop solution is added to terminate the reaction.

6. The detection method according to claim 5, characterized in that, The PCR amplification program described in step S3 is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, for a total of 40 cycles; melting curve analysis at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s.

7. The detection method according to claim 5, characterized in that, The pretreatment described in step S1 is as follows: When the sample is food, take 25g of sample and add 225mL of 0.9% sterile saline, homogenize for 2min to obtain a 1:10 homogenate, serially dilute and take 1mL for later use; when the sample is drinking water, take 10mL of sample, centrifuge at 8000r / min for 10min, discard the supernatant, and resuspend the precipitate with 1mL of sterile saline for later use; when the sample is medical sputum, add an equal volume of sterile saline, vortex to mix, centrifuge at 8000r / min for 10min, discard the supernatant and resuspend the precipitate. For medical blood samples: Take 1 mL of the sample, add 3 mL of red blood cell lysis buffer, shake for 10 min, centrifuge at 8000 r / min for 10 min, discard the supernatant and resuspend the precipitate; when the sample is environmental soil, take 10 g of the sample, add 90 mL of sterile physiological saline, shake for 30 min, let stand for 10 min, and take 1 mL of the supernatant for later use; when the sample is environmental sewage, take 10 mL of the sample, centrifuge at 8000 r / min for 10 min, discard the supernatant and resuspend the precipitate for later use.

8. The microbial detection kit according to any one of claims 1-4, characterized in that, The kit has a detection sensitivity of 1-10 CFU / mL, a specificity of 100%, a coefficient of variation of Ct value <4%, and a detection time of 70-90 min.

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