Primer pair and probe for detecting microorganisms in cow milk or cow milk drink and application of primer pair and probe

Abstract

The invention belongs to the technical field of detection of microorganisms in cow milk drinks, and particularly relates to a primer pair and a probe for detecting microorganisms in cow milk or cow milk drinks and application of the primer pair and the probe. The sequence of the primer pair is shown as follows: F: 5 '-TCCGCTAGCCAAGCAGCCG-3', and R: 5 '-CTACGGCTACCTTGTTACGACTT-3'. The invention further discloses a method for detecting the content of the The microorganisms in cow milk or cow milk drinks are detected based on the primer pair and / or probe provided by the invention, eight microorganisms in cow milk or cow milk drinks can be rapidly and accurately detected at the same time, the detection limit is low, and the sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganism detection in milk drinks, and in particular relates to a primer pair and a probe for microorganism detection in milk or milk drinks and applications thereof. Background Art

[0002] Milk or milk drinks are one of the important foods for people to obtain protein and calcium. With the improvement of living standards, the safety and sanitation of milk or milk drinks have attracted more and more attention, especially the safety and sanitation problems caused by microorganisms (such as Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes, etc.) that still exist after high-temperature sterilization due to production failures and contaminated in transportation packaging.

[0003] Traditional detection methods for microorganisms in milk or milk drinks mainly include heat preservation experiments and total colony count culture. These methods have disadvantages such as long heat preservation time, long culture time, and large manual counting errors. In addition, the detection cycle for products such as UHT (ultra-high temperature instantaneous sterilization) milk is long, which leads to increased warehouse pressure and delayed product launch. Therefore, it is necessary to develop a highly specific, sensitive and rapid method for detecting microorganisms in milk or milk drinks.

[0004] PCR (polymerase chain reaction) technology, especially fluorescent quantitative PCR technology, can achieve rapid detection of microorganisms in a short time due to its advantages such as high sensitivity, high specificity and short time consumption, providing a new idea for the detection of microorganisms in milk or milk drinks. Summary of the invention

[0005] The present invention develops a broad-spectrum specific primer and / or probe, which can achieve qualitative or quantitative detection of microorganisms in milk or milk-related drinks through PCR amplification or fluorescent real-time PCR amplification.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides a primer pair for detecting microorganisms in milk or milk beverages, and the primer pair sequence is as follows: F: 5'-TCCGCTAGCCAAGCAGCCCG-3', R: 5'-CTACGGCTACCTTGTTACGACTT-3'.

[0008] In another aspect, the present invention provides a primer pair and a probe combination for detecting microorganisms in milk or milk beverages.

[0009] The primer pair sequences are as follows:

[0010] F: 5'-TCCGCTAGCCAAGCAGCCCG-3', R: 5'-CTACGGCTACCTTGTTACGACTT-3';

[0011] The probe sequences are as follows:

[0012] 5'-TGCGGGCGTGCTTAACACATGCAAGTC-3'.

[0013] Preferably, the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group; wherein the fluorescent reporter group is Cy3, Cy5, FAM, VIC or HEX; and / or the fluorescent quencher group is TAMRA, BHQ1 or BHQ2.

[0014] In another aspect, the present invention provides a detection product for detecting microorganisms in milk or milk beverages, which comprises the primer pair of the present invention or the primer pair and probe combination of the present invention.

[0015] Preferably, the above-mentioned detection product is a PCR amplification detection reagent or a PCR amplification detection kit.

[0016] In another aspect, the present invention provides a primer pair of the present invention or a primer pair and a probe combination of the present invention for use in detecting microorganisms in milk or milk drinks, wherein the microorganisms include one or more of Escherichia coli, Staphylococcus aureus (S. aureus), Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes.

[0017] In another aspect, the present invention provides a method for detecting microorganisms in milk or milk beverages, which comprises any one of the following methods:

[0018] The first detection method comprises: (1) using the primer pair of the present invention or the detection product of the present invention to perform PCR amplification on the milk or milk drink to be detected to obtain an amplification product; (2) performing qualitative or quantitative detection of the microorganisms in the milk or milk drink based on the amplification product; or

[0019] The second detection method comprises: (I) performing PCR amplification on the milk or milk drink to be detected using the primer pair and probe combination of the present invention or the detection product of the present invention; (II) performing qualitative or quantitative detection of the microorganisms in the milk or milk drink to be detected based on the fluorescent signal;

[0020] The microorganisms include one or more of Escherichia coli, Staphylococcus aureus (S. aureus), Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes.

[0021] Preferably, the milk or milk drink is UHT sterilized milk or milk drink.

[0022] Preferably, the milk or milk drink to be tested is heated at 95° C.-98° C. for 1 min-20 min before amplification.

[0023] Preferably, the annealing temperature of the above PCR amplification is 67.4°C-67.8°C.

[0024] The beneficial effects of the present invention include:

[0025] (1) Based on the primer pairs and / or probes provided by the present invention, microorganisms in milk or milk drinks (especially UHT sterilized milk or milk drinks) are detected, and one or more microorganisms selected from Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes can be detected quickly and accurately.

[0026] (2) Based on the primer pairs and / or probes provided by the present invention, microorganisms in milk or milk drinks (especially UHT sterilized milk or milk drinks) can be detected, and eight microorganisms, including Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis and Listeria monocytogenes, can be detected simultaneously and quickly and accurately.

[0027] (3) Based on the primer pairs and / or probes provided by the present invention, microorganisms in milk or milk drinks (especially UHT sterilized milk or milk drinks) are detected, and the detection limit of microorganisms is low and the sensitivity is high. For example, the detection limit of Bacillus cereus and Bacillus subtilis can be less than 10 3 CFU / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Double distilled water conditions for different primer pairs and probe amplifications;

[0029] Figure 2 The amplification results of negative control, Escherichia coli and Staphylococcus aureus at different annealing temperatures are shown;

[0030] Figure 3 The amplification of strains by different pre-treatment methods;

[0031] Figure 4 This is a comprehensive test of the primer pairs and probes in the present invention;

[0032] Figure 5 The sensitivity test results of the primer pair and probe of the present invention to Bacillus cereus;

[0033] Figure 6 The sensitivity test results of the primer pair and probe of the present invention to Bacillus subtilis;

[0034] Figure 7 This is the reverse-linked strain test results of the primer pair and probe in the present invention. DETAILED DESCRIPTION

[0035] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0036] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0037] In the present invention, "milk drink" refers to a drink prepared by adding milk, such as walnut milk, peanut milk or strawberry milk.

[0038] The embodiment of the present invention provides a primer pair for detecting microorganisms in milk or milk beverages, and the primer pair sequence is as follows: F: 5'-TCCGCTAGCCAAGCAGCCCG-3', R: 5'-CTACGGCTACCTTGTTACGACTT-3'.

[0039] It should be noted that the primers in the present invention have strong specificity for microorganisms in milk or milk drinks (such as one or more of Escherichia coli, Staphylococcus aureus (S.aureus), Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes) and have a broad spectrum for the above-mentioned microorganisms.

[0040] The present invention also provides a primer pair and a probe combination for detecting microorganisms in milk or milk beverages.

[0041] The primer pair sequences are as follows:

[0042] F: 5'-TCCGCTAGCCAAGCAGCCCG-3',

[0043] R: 5'-CTACGGCTACCTTGTTACGACTT-3';

[0044] The probe sequences are as follows:

[0045] 5'-TGCGGGCGTGCTTAACACATGCAAGTC-3'.

[0046] It should be noted that, based on the above primer pairs, specific probes and primer pairs can also be designed for use in combination. The probe combined with the primer pair can quickly and accurately detect the above microorganisms through real-time fluorescent PCR amplification reaction.

[0047] In some specific examples, the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group; wherein the fluorescent reporter group is Cy3, Cy5, FAM, VIC or HEX; and / or the fluorescent quencher group is TAMRA, BHQ1 or BHQ2.

[0048] It should be noted that it should be understood that the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group, which is used to emit a fluorescent signal, thereby realizing the detection of the target molecule and the interaction between the fluorescent quencher group and the fluorescent reporter group to reduce the fluorescence intensity. In addition, the fluorescent reporter group and the fluorescent quencher group in the present invention can be selected from groups known in the art, such as the fluorescent reporter group can be Cy3, Cy5, FAM, VIC or HEX, and the fluorescent quencher group can be TAMRA, BHQ1 or BHQ2; in the present invention, it can be preferred that the fluorescent reporter group is FAM and the fluorescent quencher group is BHQ1, and the fluorescent reporter group and the fluorescent quencher group are more suitable for the detection in the present invention.

[0049] The embodiment of the present invention further provides a detection product for detecting microorganisms in milk or milk beverages, which includes the primer pair of the present invention or the primer pair and probe combination of the present invention.

[0050] It should be noted that the primer pairs and / or probes in the present invention can be prepared with other auxiliary detection reagents into detection products for detecting psychrophilic bacteria; wherein the auxiliary detection reagents are PCR amplification reagents known in the art, such as ddH2O reagents, DNA extraction reagents, positive controls and PCR amplification buffers, etc.

[0051] In some specific examples, the above-mentioned detection product is a PCR amplification detection reagent or a PCR amplification detection kit.

[0052] It should be noted that the detection product can be in any form that can realize detection, such as a reagent or a kit. Generally, in order to better transport and store, it is preferably prepared in the form of a detection kit, and the form of the kit is well known in the art, such as including instructions for use, containers for containing reagents, etc.

[0053] An embodiment of the present invention also provides an application of a primer pair of the present invention or a primer pair and a probe combination of the present invention in detecting microorganisms in milk or milk drinks, wherein the microorganisms include one or more of Escherichia coli, Staphylococcus aureus (S. aureus), Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes.

[0054] It should be noted that based on the specificity of the primer pairs and primer pair and probe combinations in the present invention to Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes, they can be used in the detection of microorganisms related to milk or milk drinks.

[0055] The present invention also provides a method for detecting microorganisms in milk or milk beverages, which includes any one of the following methods:

[0056] The first detection method includes:

[0057] (1) using the primer pair of the present invention or the detection product of the present invention to perform PCR amplification on the milk or milk beverage to be tested to obtain an amplification product;

[0058] (2) qualitative or quantitative detection of microorganisms in milk or milk beverages based on the amplified products; or

[0059] The second detection method includes:

[0060] (I) performing PCR amplification on the milk or milk beverage to be tested using the primer pair and probe combination of the present invention or the detection product of the present invention;

[0061] (II) qualitatively or quantitatively detecting microorganisms in the milk or milk beverage to be detected based on the fluorescent signal;

[0062] The microorganisms include one or more of Escherichia coli, Staphylococcus aureus (S. aureus), Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes.

[0063] It should be noted that in the first detection method mentioned above, after amplification by the primer pair of the present invention, the amplification product is analyzed, thereby realizing the detection of Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes; the method for analyzing the amplification product includes conventional means such as high-throughput testing and gel electrophoresis.

[0064] It should also be noted that in the above-mentioned second detection method, amplification is carried out through the probe and primer pairs, and the amplification reaction can be detected according to the real-time fluorescence during amplification, thereby realizing qualitative or quantitative detection of Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes.

[0065] In some specific examples, the milk or milk beverage is UHT sterilized milk or milk beverage.

[0066] It should be noted that the present invention has verified that no nucleic acid of microorganisms such as Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes will remain in UHT sterilized milk or milk drinks, which will not affect the detection of the above microorganisms and can ensure the accuracy of the detection.

[0067] In some specific examples, the milk or milk beverage to be tested is heated at 95° C.-98° C. for 1 min-20 min before amplification.

[0068] It should be noted that different pre-treatment methods of the samples to be tested have an impact on the PCR amplification test results. In the present invention, it is preferred to perform pre-treatment by heating at 95°C-98°C for 1min-20min, and its subsequent amplification effect is better than that of the lysis solution treatment method and the physiological saline treatment method; the heating temperature can be 95.5°C, 96°C, 96.5°C, 97°C or 97.5°C, etc., and the treatment time can be 5min, 7min, 10min, 15min, 17min or 19min, etc.

[0069] In some specific examples, the annealing temperature of the above PCR amplification is 67.4°C-67.8°C.

[0070] It should be noted that the annealing temperature has a certain influence on the accuracy of PCR amplification detection. In the present invention, 67.4°C-67.8°C is preferred, and 67.4°C is more preferred. Between 67.4°C-67.8°C, the CT value of the microorganisms (such as Escherichia coli and Staphylococcus aureus) detected in the present invention is within 40, and the CT value of the negative control is close to 40; at 67.4°C, the CT values ​​of the microorganisms (such as Escherichia coli and Staphylococcus aureus) detected in the present invention are the smallest, and the CT value of the negative control is close to 40. It is shown that under the above annealing temperature treatment, the primer pairs and probes in the present invention have stronger specificity for organisms, and the detection is more sensitive and accurate.

[0071] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0072] In the following examples, Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Citrobacter freundii CICC 25015, Bacillus cereus CICC 23828, Salmonella ATCC22542, Bacillus subtilis CICC 10027, Enterococcus faecalis CICC 25019 and Listeria monocytogenes CICC 21633 were purchased from China Microorganism Collection Center and Wuhan Microorganism Collection Center.

[0073] 1. Primer and probe screening

[0074] (I) Design of primers and probes

[0075] Based on the conserved sequence of bacterial 16S rRNA gene, a primer pair and probe combination targeting broad spectrum of bacteria was designed. The sequence of the primer and probe combination is as follows:

[0076] The first pair of primers and probes (also called primer 1, the same below):

[0077] Forward primer (F1): 5'-TGCGGGCGTGCTTAACAC-3',

[0078] Reverse primer (R1): 5'-GGTAGCCCTGCTTGGCTAGC-3',

[0079] Probe (P1): 5′-FAM-TGCAAGCTGCGGGCGTGC-BHQ1-3′;

[0080] The second pair of primers and probes (also called primer 2, the same below):

[0081] Forward primer (F2): 5'-CAGCCCGCTTAGCGTAG-3',

[0082] Reverse primer (R2): 5'-ACTCGTAACAAGGTAGCCG-3',

[0083] Probe (P2): 5′-FAM-CGTAGCGCTTAGCGGGCTG-BHQ1-3′;

[0084] The third pair of primers and probes (also referred to as primer 3, the same below):

[0085] Forward primer (F3): 5'-CCGCTAGCCAAGCAG-3',

[0086] Reverse primer (R3): 5'-GTCGTAACAAGGTAGCC-3',

[0087] Probe (P3): 5′-FAM-CAGCCGCTTAGCGTAGC-BHQ1-3′;

[0088] The fourth pair of primers and probes (also referred to as primer 4, the same below):

[0089] Forward primer (F4): 5'-TTGGCGTGCTTAACACA-3',

[0090] Reverse primer (R4): 5'-GTACCTGGTTACGACTCAC-3',

[0091] Probe (P4): 5′-FAM-TTAACACATGCAAGTCGG-BHQ1-3′;

[0092] The fifth pair of primers and probes (also referred to as primer 5, the same below):

[0093] Forward primer (F5): 5'-GTGCTTAACACATGCAA-3',

[0094] Reverse primer (R5): 5'-GACTCGTTAACCAGGTAG-3',

[0095] Probe (P5): 5′-FAM-ACATGCAAGTCGGTAGCC-BHQ1-3′;

[0096] The sixth pair of primers and probes (also referred to as primer 6, the same below):

[0097] Forward primer (F6): 5'-CTAACACATGCAAGTCG-3',

[0098] Reverse primer (R6): 5'-GGTTAGCGTAGCGCTTA-3',

[0099] Probe (P6): 5′-FAM-GCAAGTCGGTAGCCCTGC-BHQ1-3′;

[0100] The seventh pair of primers and probes (also referred to as primer 7, the same below):

[0101] Forward primer (F7): 5'-CATGCAAGTCGGTAGCC-3',

[0102] Reverse primer (R7): 5'-TAGCGCTTAGCGGGCGT-3',

[0103] Probe (P7): 5′-FAM-AAGTCGGTAGCCCTGCTT-BHQ1-3′;

[0104] The eighth pair of primers and probes (also referred to as primer 8, the same below):

[0105] Forward primer (F8): 5'-TGCAGGCCGCCTTAGC-3',

[0106] Reverse primer (R8): 5'-AGGCCAGGCTGCCGTCTACGG-3',

[0107] Probe (P8): 5′-FAM-GCCTGGACCGGTCCAGCTG-BHQ1-3′;

[0108] The ninth pair of primers and probes (also referred to as primer 9, the same below):

[0109] Forward primer (F9): 5'-TCCGCTAGCCAAGCAGCCCG-3',

[0110] Reverse primer (R9): 5'-CTACGGCTACCTTGTTACGACTT-3', probe (P9): 5'-FAM-TGCGGGCGTGCTTAACACATGCAAGTC-BHQ1-3'.

[0111] (II) Screening of primer pairs and probe combinations

[0112] The above 9 pairs of different primers and probes were screened by using double distilled water, wherein the fluorescence quantitative PCR amplification procedure and system are shown in Table 1 below.

[0113] Table 1 Fluorescence quantitative PCR amplification procedures and systems

[0114]

[0115] Fluorescence quantitative PCR amplification Figure 1 As shown in the results, only primer 9 did not initiate the PCR, while the other 8 primer pairs all initiated the PCR to varying degrees, so primer 9 was selected as the primer for subsequent experiments.

[0116] 2. PCR amplification program optimization test

[0117] Optimizing the annealing temperature in PCR is the key to improving the specificity and efficiency of the reaction. A higher annealing temperature can reduce nonspecific binding, reduce the formation of primer dimers and nonspecific products, and thus improve the specificity of PCR; however, too low an annealing temperature may lead to increased sensitivity but reduced specificity. Therefore, optimizing the annealing temperature can improve specificity while maintaining sensitivity. In addition, the characteristics of different primers and templates and other conditions in the PCR reaction will also affect the selection of the optimal annealing temperature. By adjusting the annealing temperature, the effective binding of primers to template DNA can be ensured, the success rate of the PCR reaction can be improved, and more reliable and accurate PCR results can be obtained.

[0118] In this experiment, the annealing temperature of primer 9 was optimized by using Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 (the above strains were purchased from China Microbiological Culture Collection Center), specifically including: Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were cultured in solid and liquid respectively for 10 7 CFU / mL, and then according to the fluorescence quantitative PCR amplification procedure and system in Table 1 (10 7 CFU / mL bacterial solution as DNA template) to amplify Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923; double distilled water ddH2O was set as a negative control; wherein the annealing temperatures were set to 67.0℃, 67.4℃, 67.8℃, 68.6℃, 69.6℃, 70.4℃, 70.8℃ and 71.0℃ respectively; the CT values ​​of the negative control, Escherichia coli and Staphylococcus aureus at different annealing temperatures were detected, and the results were as follows Figure 2 and as shown in Table 2 below.

[0119] Table 2 CT values ​​of negative control, Escherichia coli and Staphylococcus aureus at different annealing temperatures

[0120] Annealing temperature CT value of negative control CT value of Escherichia coli CT value of Staphylococcus aureus 67.0℃ 36.63 20.47 20.5 67.4℃ 38.31 20.51 20.67 67.8℃ 39.21 20.55 22.74 68.6℃ 39.67 23.69 31.27 69.6℃ 2.22 36.74 39.6 70.4℃ 1.28 N / A 39.62 70.8℃ N / A N / A N / A 71.0℃ N / A N / A N / A

[0121] From the above table 2 and Figure 2 It can be seen that when the annealing temperature is 67.4°C, the CT value of the positive control strain is the lowest, and the CT value of the negative control is close to 40. Therefore, 67.4°C can be preferentially selected as the annealing temperature in the future.

[0122] 3. Optimization experiment of pre-amplification treatment of UHT sterilized milk

[0123] Optimization of PCR pre-treatment aims to improve the overall performance of the PCR reaction. By carefully designing primers, ensuring the quality and concentration of template DNA, and possible other pre-treatment steps, the specificity of PCR can be significantly enhanced and the interference of nonspecific amplification can be reduced. At the same time, these optimization measures can also improve the efficiency of PCR, making the reaction faster and more sensitive. Therefore, optimization of PCR pre-treatment is an important part of ensuring the accuracy and reliability of the experiment. It directly affects the accuracy of subsequent data analysis and the reliability of biological conclusions, and is an indispensable part of molecular biology research.

[0124] The bacterial solution is Escherichia coli cultured overnight, with a concentration of about 10 8 CFU / mL or so.

[0125] In this experiment, the effects of three UHT sterilized milk pre-amplification treatments on amplification detection were compared, as follows:

[0126] In the first treatment, UHT sterilized milk was inoculated with E. coli ATCC25922 and cultured until the concentration of E. coli reached 10 8 CFU / mL, then the UHT sterilized milk containing E. coli was heated to 98°C and kept for 10 min, and then PCR amplification was performed according to the fluorescence quantitative PCR amplification procedure and system in Table 1 (UHT sterilized milk containing E. coli was used as DNA template), wherein the annealing temperature was set to 67.4°C, and the CT value was detected;

[0127] The second treatment method: inoculate UHT sterilized milk with E. coli ATCC25922 and culture until the concentration of E. coli is 10 8 CFU / mL, then the UHT sterilized milk containing Escherichia coli was treated with lysis buffer (purchased from Shandong Meizheng, the volume ratio of UHT sterilized milk to lysis buffer was 1:1) for 2 h, and PCR amplification was performed according to the fluorescence quantitative PCR amplification procedure and system in Table 1 above (UHT sterilized milk containing Escherichia coli was used as DNA template), wherein the annealing temperature was set to 67.4 °C, and the CT value was detected;

[0128] The third treatment method: inoculate UHT sterilized milk with E. coli ATCC25922 and culture until the concentration of E. coli is 10 8 CFU / mL, then the UHT sterilized milk containing E. coli was added to normal saline (mass fraction 0.85% normal saline, volume ratio of UHT sterilized milk to normal saline was 1:1), and PCR amplification was performed according to the fluorescence quantitative PCR amplification procedure and system in Table 1 above, wherein the annealing temperature was set to 67.4°C, and the CT value was detected;

[0129] The results are as follows Figure 3As shown, the results show that at the same bacterial concentration (10 8 CFU / mL), the CT value of heat treatment is the smallest, so the pretreatment in the present invention can preferably be heat treatment at 98°C for 10min.

[0130] IV. Extensive testing of primer pairs and probes

[0131] The reason for the wide adaptability of PCR is its unique advantages and wide application fields; PCR technology has high sensitivity and specificity, and can accurately amplify and detect trace amounts of target DNA or RNA sequences, which is the basis of its wide adaptability. By verifying the wide adaptability of PCR, it can be ensured that it can maintain stable performance in different sample types, different pathogens and different experimental conditions.

[0132] In this experiment, Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Citrobacter freundii CICC 25015, Bacillus cereus CICC 23828, Salmonella ATCC22542, Bacillus subtilis CICC 10027, Enterococcus faecalis CICC 25019, Listeria monocytogenes CICC 21633, and laboratory-preserved Klebsiella, Streptococcus agalactiae, Streptococcus dysgalactiae, and Staphylococcus epidermidis were cultured in solid and liquid culture to a concentration of 10 7 CFU / mL, and then heat treated at 98°C for 10 min. Then, PCR amplification was performed according to the fluorescence quantitative PCR amplification procedure and system in Table 1 (bacterial solution was used as DNA template), where the annealing temperature was set to 67.4°C, and the fluorescence of different strains was detected; at the same time, double distilled water (ddH2O) was set as a negative control.

[0133] Test results Figure 4 As shown, the results show that all the bacteria tested can be detected with good curves, indicating that the primer pairs and probes in the present invention have good wide adaptability.

[0134] V. Sensitivity test of primer pairs and probes

[0135] In terms of rapid bacterial detection, the sensitivity verification of fluorescent quantitative PCR is crucial. First, high sensitivity means that fluorescent quantitative PCR can accurately detect extremely low concentrations of bacterial DNA, which is of great significance for the early detection and diagnosis of bacterial infections; secondly, sensitivity verification can ensure the accuracy and reliability of experimental results, avoid the occurrence of false negative or false positive results, and thus guide doctors to formulate correct treatment plans. In addition, through sensitivity verification, experimental conditions can be optimized, detection efficiency can be improved, and detection time can be shortened, making fluorescent quantitative PCR more advantageous in rapid bacterial detection.

[0136] In this experiment, the final concentration of overnight culture was 10 6 Bacillus cereus CICC23828 and Bacillus subtilis CICC 10027 with CFU / mL were used as positive strains (these two Bacillus species are difficult to detect and are the main strains contaminated in dairy products), and double distilled water (ddH2O) was set as a negative control; the concentrations of Bacillus cereus and Bacillus subtilis were diluted to 10 5 CFU / mL, 10 4 CFU / mL and 10 3 CFU / mL; then PCR amplification was performed according to the fluorescence quantitative PCR amplification procedure and system in Table 1 above (bacterial solution was used as DNA template), wherein the annealing temperature was set to 67.4°C, and the fluorescence of different strains was detected.

[0137] The results are as follows Figure 5 and Figure 6 As shown (in the figure, 10 6 , 10 5 , 10 4 and 10 3 Refers to 10 respectively 6 CFU / mL 10 5 CFU / mL, 10 4 CFU / mL and 10 3 CFU / mL), the results showed that the detection limit of Bacillus cereus CICC 23828 and Bacillus subtilis CICC 10027 was 10 3 CFU / mL, which can meet the testing requirements of the product after insulation.

[0138] 6. Microbial counter-inoculation test

[0139] In this test, a total of 12 products of different types (all from Chongqing Tianyou Dairy Co., Ltd.) were subjected to reverse inoculation test. The indicator strains of reverse inoculation were Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Citrobacter freundii CICC 25015, Bacillus cereus CICC 23828, Salmonella ATCC22542, Bacillus subtilis CICC 10027, Enterococcus faecalis CICC 25019 and Listeria monocytogenes CICC 21633. After overnight culture, the above bacteria were added to 12 products separately or mixedly, and the final concentration of the separate culture was 1-10 CFU / mL, and the final concentration of the mixed culture was 8×(1-10) CFU / mL; then, PCR amplification detection was performed according to the fluorescence quantitative PCR amplification procedure and system in Table 1 (the bacterial solution was used as a DNA template), wherein the annealing temperature was set to 67.4°C; the detection results are shown in Tables 3 and 4.

[0140] Table 3 Single microorganism counter-inoculation test results

[0141]

[0142] Table 4 Mixed microbial counter-inoculation test results

[0143]

[0144]

[0145] It can be seen from Tables 3 and 4 above that 8 strains inoculated with 1-10 CFU / mL in 12 products can all be detected, and 8 strains in mixed strains can also be detected simultaneously.

[0146] VII. Nucleic acid residue test

[0147] The impact of high temperature on nucleic acids is mainly reflected in its ability to cause nucleic acid denaturation; as the temperature rises, the hydrogen bonds in the secondary structure of nucleic acids will gradually break, causing the originally tight double-stranded structure to become loose, such as the double-stranded structure of DNA will separate into single strands.

[0148] In the test of the present invention, in order to detect whether there are nucleic acid residues of Escherichia coli, Staphylococcus aureus, Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis and Listeria monocytogenes in milk after UHT sterilization, thereby affecting the accuracy of subsequent detection; Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Citrobacter freundii CICC 25015, Bacillus cereus CICC 23828, Salmonella ATCC22542, Bacillus subtilis CICC 10027, Enterococcus faecalis CICC 25019 and Listeria monocytogenes CICC 21633 are used as indicator strains, and they are respectively cultured in solid and liquid to a concentration of 10 7 CFU / mL (i.e., the original bacterial suspension), and then sterilized at 121°C for 15 minutes; then, according to the fluorescence quantitative PCR amplification procedure and system in Table 1 above (the bacterial suspension was used as a DNA template, and the annealing temperature was set to 67.4°C), the original bacterial suspension and the sterilized bacterial suspension were subjected to PCR amplification and CT value testing; the test results are shown in Tables 5 and Figure 7 shown.

[0149] Table 5 CT values ​​of different indicator strains before and after high temperature

[0150]

[0151] From Table 5 and Figure 7It can be seen that after high-temperature sterilization, the CT values ​​of 8 foodborne pathogens are all around 40, that is, the bacteria before high-temperature sterilization will not affect the detection of bacteria contaminated in the product later.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A primer pair for detecting microorganisms in milk or milk beverages, characterized in that: The primer pair sequences are as follows: F: 5'-TCCGCTAGCCAAGCAGCCCG-3', R: 5'-CTACGGCTACCTTGTTACGACTT-3'.

2. A primer pair and probe combination for detecting microorganisms in milk or milk beverages, characterized in that: The primer pair sequences are as follows: F: 5'-TCCGCTAGCCAAGCAGCCCG-3', R: 5'-CTACGGCTACCTTGTTACGACTT-3'; The probe sequences are as follows: 5'-TGCGGGCGTGCTTAACACATGCAAGTC-3'.

3. The primer pair and probe combination according to claim 2, characterized in that: The 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group; wherein the fluorescent reporter group is Cy3, Cy5, FAM, VIC or HEX; and / or the fluorescent quencher group is TAMRA, BHQ1 or BHQ2.

4. A detection product for detecting microorganisms in milk or milk drinks, characterized in that: It comprises the primer pair according to claim 1 or the primer pair and probe combination according to claim 2 or 3.

5. The detection product according to claim 4, characterized in that: The detection product is a PCR amplification detection reagent or a PCR amplification detection kit.

6. Use of the primer pair according to claim 1 or the primer pair and probe combination according to claim 2 or 3 in detecting microorganisms in milk or milk drinks, wherein the microorganisms include one or more of Escherichia coli, Staphylococcus aureus (S. aureus), Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes.

7. A method for detecting microorganisms in milk or milk beverages, characterized in that: Choose one of the following methods: The first detection method includes: (1) using the primer pair described in claim 1 or the detection product described in claim 4 or 5 to perform PCR amplification on the milk or milk drink to be tested to obtain an amplification product; (2) qualitative or quantitative detection of microorganisms in milk or milk beverages based on the amplified products; or the second detection method includes: (I) performing PCR amplification on the milk or milk beverage to be tested using the primer pair and probe combination of claim 2 or 3 or the detection product of claim 4 or 5; (II) qualitatively or quantitatively detecting microorganisms in the milk or milk beverage to be detected based on the fluorescent signal; The microorganisms include one or more of Escherichia coli, Staphylococcus aureus (S. aureus), Citrobacter freundii, Bacillus cereus, Salmonella, Bacillus subtilis, Enterococcus faecalis or Listeria monocytogenes.

8. The detection method according to claim 7, characterized in that: The milk or milk beverage is UHT sterilized milk or milk beverage.

9. The detection method according to claim 7 or 8, characterized in that: The milk or milk drink to be tested is heated at 95°C-98°C for 1 min-20 min before amplification.

10. The detection method according to claim 7 or 8, characterized in that: The annealing temperature for PCR amplification is 67.4°C-67.8°C.

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