CN114990242A

CN114990242A - PCR amplification primer pair and application thereof

Info

Publication number: CN114990242A
Application number: CN202210706287.4A
Authority: CN
Inventors: 张凤; 祁腾; 张清平; 王静; 唐甜; 舒希; 朱旭
Original assignee: Chongqing Tianyou Dairy Co Ltd
Current assignee: Chongqing Tianyou Dairy Co Ltd
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2022-09-02
Also published as: LU502570B1

Abstract

The invention belongs to the technical field of bacteria detection, and particularly relates to a PCR amplification primer pair and application thereof, which are particularly suitable for detecting heat-resistant bacillus. The upstream primer of the PCR amplification primer pair comprises the nucleotide sequence shown as SEQ ID NO: 1, and the downstream primer comprises a sequence shown as SEQ ID NO: 2. When the PCR amplification primer pair is used for detecting heat-resistant bacillus, the specificity is strong, and the PCR amplification primer pair can effectively perform specific amplification with the heat-resistant bacillus; when the real-time fluorescence PCR amplification detection method based on the PCR amplification primer pair is used for detecting the heat-resistant bacillus, the detection can be completed within about 90min, and the detection time is short; the detection limit for detecting the heat-resistant bacillus in the cow milk can reach 11CFU/mL, and the sensitivity is high.

Description

PCR amplification primer pair and application thereof

Technical Field

The invention belongs to the technical field of bacteria detection, and particularly relates to a PCR amplification primer pair and application thereof, which are particularly suitable for detecting heat-resistant bacillus.

Background

Bacillus thermostabilis (BSTD) is a group of endospore-producing bacteria with high heat resistance. In the 19 th century 80 modern times, BSTD was found in ultra high temperature sterilized (UHT) milk in germany, and subsequently, this bacterium was also found in succession in other countries in europe. BSTD was studied and named by Bertil in 1996, and the species is of the kingdom bacteria, the phylum firmicutes, the class Bacillales, the order Bacillales, the family Baciliaceae, the genus Bacillus, the species BSTD; the model strain number of this strain was M215(DSMZ 10599).

BSTD may survive and propagate in some food or environment, and as the bacillus may not change the quality and taste of food obviously and has no pathogenicity, it is easy to ignore in microbe detection. Particularly in dairy products, as cow milk is the best culture medium for bacterial reproduction, the probability of bacillus and spores thereof appearing in raw milk is high; the study by Hammer et al shows that the milk contains BSTD up to 10 after being cultured at 30 ℃ for 5 days ⁵ CFU/mL. Although BSTD can not cause dairy product deterioration and has no pathogenicity, the BSTD can directly influence the quality of the dairy product, and can reflect that flaws exist in the production process of the product and unknown risks also exist; moreover, the national new national standard requirement for microorganisms in cow milk is not higher than 200 ten thousand per milliliter, so that BSTD needs to be comprehensively and deeply known, and a more perfect microorganism control system is established for dairy enterprises.

In the above, food enterprises, especially dairy enterprises, need to monitor BSTD in real time to realize effective prevention and control of bacillus in industrial production, so an effective method for detecting heat-resistant bacillus is necessary.

Disclosure of Invention

In order to solve the above problems, it is an object of the present invention to provide a pair of PCR amplification primers having strong specificity to Bacillus thermotolerans, by which the Bacillus thermotolerans can be qualitatively and quantitatively analyzed by a PCR amplification detection method.

In order to achieve the purpose, the following technical scheme can be adopted:

in one aspect, the present invention provides a PCR amplification primer pair, wherein the upstream primer can comprise a primer as shown in seq id no: 1, the downstream primer may comprise a sequence as shown in seq id no: 2.

In another aspect, the invention provides a detection reagent, which may include the PCR amplification primer pair described above.

In still another aspect, the invention provides a detection kit, which may include the PCR amplification primer pair or the detection reagent.

The invention further provides an application of the PCR amplification primer pair or the detection reagent or the detection kit in detection of heat-resistant bacillus.

In another aspect, the present invention provides a method for detecting the PCR amplification of bacillus thermotolerans, comprising: and carrying out PCR amplification on the DNA template to be detected by using the PCR amplification primer pair to obtain a PCR amplification product, and analyzing the PCR amplification product so as to judge whether the DNA template to be detected contains heat-resistant bacillus.

In another aspect, the present invention provides a method for detecting thermophilic bacillus by PCR amplification, comprising: mixing the DNA template to be detected with the detection reagent, and then carrying out PCR amplification, wherein in the PCR amplification process, whether the DNA template to be detected contains heat-resistant bacillus or not is judged according to a fluorescent signal; and judging the content of the heat-resistant bacillus in the DNA template to be detected according to the CT value and the standard curve.

The beneficial effects of the invention at least comprise:

(1) the PCR amplification primer pair provided by the invention has strong specificity when detecting heat-resistant bacillus, can effectively perform specific amplification with the heat-resistant bacillus, and cannot generate false positive;

(2) when the real-time fluorescence PCR amplification detection method based on the PCR amplification primer pair provided by the invention is used for detecting heat-resistant bacillus, the detection can be completed within about 90min, and the detection time is short;

(3) the detection limit of the real-time fluorescence PCR amplification detection method based on the PCR amplification primer pair for detecting the heat-resistant bacillus in the culture medium can reach 1 CFU/mL; the detection limit for detecting the heat-resistant bacillus in the cow milk can reach 11CFU/mL, and the sensitivity is high.

Drawings

FIG. 1 is a microscopic examination of activated heat-resistant Bacillus bovis B1;

FIG. 2 is a phylogenetic tree of B1B;

FIG. 3 shows the specific detection of different bacteria by the primers in the present example;

FIG. 4 shows the specific detection of bacteria for different pairs of primers in different embodiments of the present invention;

FIG. 5 is a graph showing the effect of different annealing temperatures on the detection sensitivity of B1;

FIG. 6 shows the detection limit of Bacillus thermotolerans B1 in liquid medium for primer pairs in the examples of the present invention;

FIG. 7 is a standard curve of Bacillus thermotolerans B1 in liquid medium for primer pairs in examples of the present invention;

FIG. 8 shows the effect of different pretreatment methods on the specificity of primers in the present example;

FIG. 9 shows the effect of different milk sample additions on the detection of B1;

FIG. 10 shows the effect of different annealing temperatures on the quantitative detection of the fluorescence of B1-Thermobacillus thermotolerans in cow's milk.

FIG. 11 shows the detection limit of the primers for Bacillus thermotolerant B1 in cow's milk in accordance with the present invention;

FIG. 12 is a standard curve of primers against B1B in bovine milk according to an embodiment of the present invention.

Detailed Description

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context has a significantly different meaning, the singular forms of expressions include the plural forms of expressions. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to refer to the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. 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 be present or may be added. As used herein, "/" can be interpreted as "and" or "depending on the circumstances.

In one aspect of the invention, a PCR amplification primer pair is provided, and in some embodiments, the upstream primer can comprise a primer sequence as shown in seq id no: 1, the downstream primer may comprise a sequence as shown in seq id no: 2.

Specifically, heat-resistant bacillus is separated and screened from milk, 16srDNA sequencing is carried out on the bacillus, and the sequence is compared with the 16srDNA of a standard mode strain M215, so that the homology reaches 100%; designing PCR amplification Primer pairs for the separated and selected heat-resistant bacillus through DNAman and Primer6 so as to obtain the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, which has strong specificity to the separated heat-resistant bacillus.

In another aspect, the invention provides a detection reagent, and in some embodiments, the detection reagent comprises at least the PCR amplification primer pair described above. Specifically, the PCR amplification primer pair can be combined with a conventional auxiliary reagent to form a detection reagent for detecting heat-resistant bacillus; conventional auxiliary reagents are known in the art, such as PCR reaction buffer, DNA polymerase or dNTP in a PCR amplification reaction system. In the above, the detection reagent comprising the PCR amplification primer pair can allow bacillus thermotolerans to be specifically amplified with the PCR amplification primer pair in the detection reagent.

Further, the detection reagent may further include a labeling reagent, and the labeling reagent includes, but is not limited to, a fluorescent dye or a fluorescent probe. Specifically, the detection efficiency and sensitivity can be further improved by adding a labeling reagent into the detection reagent, for example, a fluorescent dye or a fluorescent probe is added into a PCR amplification reaction system, the fluorescent dye or the fluorescent probe can label the synthesized double strand during the formation of the double strand in the PCR amplification, and the change of a fluorescent signal can be monitored by some external devices, such as a PCR fluorescence detector, so that the PCR amplification condition can be detected in real time.

It should be noted that the fluorescent dye and the fluorescent probe each have advantages and disadvantages. The fluorescent dye has the advantages of convenient use, no need of designing a complex probe, relatively low price and the like, but also has the defects of no template specificity, higher requirement on primer specificity, incapability of carrying out multiple quantification, relatively low sensitivity and the like; the fluorescent probe has the advantages of high specificity, good repeatability, high sensitivity, capability of performing multiple quantification and the like, but is only suitable for a specific target, relatively high in price and difficult to find the defects of low background and the like. In the detection, a fluorescent dye or a fluorescent probe can be selected and used according to the specific situation.

Further, the fluorescent dye may be a fluorescent dye known in the art, such as SYBRGreen; the fluorescent probe may be designed by itself according to a method known in the art, or may be designed by a design company.

In yet another aspect, the invention provides a detection kit, and in some embodiments, the detection kit may include the PCR amplification primer pair described above or the detection reagent described above. Specifically, the detection kit is composed in a conventional form known in the art, for example, the detection kit is provided with a kit instruction, for example, a reagent bottle containing various detection reagents is provided, for example, a plurality of small lattices are provided for placing the detection reagent bottle, for example, a dropper is provided for removing the detection reagent and the like. Similarly, based on the strong specificity of the PCR amplification primers in the detection kit to Bacillus thermotolerans, the detection kit can also effectively perform qualitative and quantitative analysis on Bacillus thermotolerans.

The invention further provides an application of the PCR amplification primer pair or the detection reagent or the detection kit in detection of heat-resistant bacillus. Specifically, when bacillus thermotolerans is amplified by using the PCR amplification primer pair or the detection reagent, the bacillus thermotolerans can be specifically amplified; when the detection reagent containing the fluorescent reagent or the fluorescent probe is used for amplification, stronger fluorescence can be generated in the amplification process, and detection and analysis are facilitated.

The PCR amplification primers or the detection reagent can be combined with a microfluidic chip to be applied to detection of the heat-resistant bacillus, and the characteristics of high efficiency and convenience of the microfluidic chip are combined, so that the detection of the heat-resistant bacillus is more convenient.

In another aspect, the present invention provides a method for detecting bacillus thermotolerans by PCR amplification, which may include: and carrying out PCR amplification on the DNA template to be detected by using the PCR amplification primer pair to obtain a PCR amplification product, and analyzing the PCR amplification product so as to judge whether the DNA template to be detected contains heat-resistant bacillus.

Specifically, in the PCR amplification detection method for bacillus thermotolerans, the bacillus thermotolerans in the DNA template to be detected can be qualitatively analyzed by detecting the PCR amplification product. Gel electrophoresis is generally selected to be performed on the PCR amplification product, and it should be understood that the amount of the PCR amplification product is an amount sufficient for gel electrophoresis, for example, 10 cycles of amplification are sufficient for gel electrophoresis, and then 10 cycles of amplification product are subjected to gel electrophoresis, for example, 20 cycles of amplification are required for gel electrophoresis, and then 20 cycles of amplification product are subjected to gel electrophoresis; the method for performing gel electrophoresis on the product after PCR amplification may be any method known to those skilled in the art, for example, the size and position of the electrophoretic target band are compared with a marker, so as to perform qualitative analysis on the DNA template to be detected, and determine whether the DNA template to be detected contains bacillus thermotolerant; relative content of the target band can also be analyzed by using imagej, so that quantitative analysis of products after PCR amplification is realized. The PCR amplification detection method for the heat-resistant bacillus can judge whether the DNA template to be detected contains the heat-resistant bacillus more accurately based on the strong specificity of the PCR amplification primer pair to the heat-resistant bacillus.

In another aspect, the present invention provides a method for detecting bacillus thermotolerans by PCR amplification, which may include: mixing the DNA template to be detected with the detection reagent, and then carrying out PCR amplification, wherein in the PCR amplification process, whether the DNA template to be detected contains heat-resistant bacillus or not is judged according to a fluorescent signal; and judging the content of the heat-resistant bacillus in the DNA template to be detected according to the CT value and the standard curve.

Specifically, in addition to the method of performing conventional PCR amplification detection using the PCR amplification primer pair and then performing quantitative and qualitative analysis on the final product of PCR amplification by using gel electrophoresis, the detection reagent can be used for performing fluorescence PCR amplification detection, i.e. introducing a labeling reagent into PCR amplification to label the formed DNA double strand during annealing and extension; therefore, the PCR amplification process can be monitored in real time through a fluorescent signal emitted by the labeling reagent, and the qualitative and quantitative analysis can be carried out on the DNA template to be detected. It should be noted that, the method for qualitatively and quantitatively analyzing the DNA template to be detected may be any method known in the art, for example, the method may be performed by qualitatively analyzing the DNA template to be detected according to whether the fluorescence-labeled amplification curve is on line, and the on line indicates that the DNA template to be detected contains bacillus thermotolerant; for example, a threshold value can be set, then a CT value is obtained, and the content of the heat-resistant bacillus in the DNA template to be detected is obtained according to the relation between the CT value and the standard curve. It is understood that a standard curve refers to a curve that is detected by a standard of known copy number, and is within the skill of the art. Similarly, based on the strong specificity of the PCR amplification primer pair to the heat-resistant bacillus, the PCR amplification detection method for the heat-resistant bacillus can also accurately perform qualitative and qualitative analysis on the DNA template to be detected.

The PCR amplification detection using the PCR amplification primer pair is different from the PCR amplification detection using the detection reagent. The PCR amplification detection using the PCR amplification primer pair needs gel electrophoresis and may need pre-enrichment, the overall detection time is longer, more than 72 hours, and only the final PCR product can be analyzed, the analysis result can only carry out qualitative analysis on the DNA template to be detected, and cannot carry out quantitative analysis on the DNA template to be detected; the detection reagent (comprising the PCR amplification primer pair and the labeling reagent) is used for PCR amplification detection, gel electrophoresis is not needed, the detection time is about 90min, and the detection time is relatively short; the annealing and extension processes in the PCR process can be monitored in real time through the fluorescent signals, the overall condition of the PCR amplification process can be integrally mastered, and the method is more favorable for research; and can also accurately perform qualitative and quantitative analysis on the DNA template to be detected. In the above, when detecting Bacillus thermotolerans, the two different PCR amplification detection methods can be selected according to actual needs.

Further, the annealing temperature for PCR amplification detection can be 56-60 ℃. Specifically, the annealing temperature mainly depends on the base composition, length and concentration of the primer; the proper annealing temperature is especially critical to the specificity of the PCR amplification reaction. Generally, the effect of the high or low annealing temperature affects the specificity and sensitivity (amplification efficiency) of the PCR reaction, and the two are inversely related, i.e., a lower annealing temperature can increase the sensitivity but has a lower specificity, while a higher annealing temperature can increase the specificity but decreases the amplification efficiency. The annealing temperature set at 56-60 deg.C, such as 57 deg.C, 58 deg.C or 59 deg.C, can make the above-mentioned PCR amplification primer pair maintain higher specificity in the above-mentioned PCR amplification reaction, and also can make the PCR amplification reaction rate maintain higher level.

Further, the concentration of the DNA template to be detected may be: the concentration of the DNA template to be detected is more than 0.5 mu L/20 mu L and less than 2 mu L/20 mu L; the concentration of the DNA template to be detected has certain influence on the PCR amplification efficiency, and has certain influence on both the fluorescence intensity and the CT value if labeled by using a labeling reagent in the PCR amplification, and in the invention, the concentration of the DNA template to be detected is between 0.5 muL/20 muL and 2 muL/20 muL, such as 1 muL/20 muL, 1.5 muL/20 muL or 1.7 muL/20 muL; under the concentration of the DNA template to be detected, the fluorescence intensity in the PCR amplification process is stronger, the CT value is less, and the detection is more facilitated.

It will be appreciated that thermophilic bacillus species are typically present in food products and it is necessary to first isolate the population of microorganisms from the food product, prepare the DNA template to be detected and then analyze it using the PCR amplification detection method for thermophilic bacillus species described above. After the content of the heat-resistant bacillus in the DNA template to be detected is obtained through analysis, the content of the heat-resistant bacillus in the food can be calculated according to the concentration of the DNA template to be detected, so that the aim of detecting the content of the heat-resistant bacillus in the food is fulfilled. It is noted that the method of isolating the microbial population from the food product is a method known in the art.

Further, the heat-resistant bacillus is heat-resistant bacillus in cow milk, and before PCR amplification detection, the cow milk to be detected is pretreated, and the pretreatment method can comprise the following steps: (1) centrifuging the milk to be detected to remove supernatant and fat, and filtering to remove the milk completely; (2) carrying out isometric resuspension by using sterile pure water, and centrifuging to retain supernatant; (3) the supernatant is subjected to metal bath at 95-105 ℃, quick-frozen and freeze-thawed. Specifically, the cow milk contains some inhibitors capable of inhibiting PCR amplification reaction, the cow milk needs to be pretreated before PCR amplification, different treatment modes have different degrees of influence on the PCR amplification process, and the treatment modes can reduce the inhibitors, so that the fluorescence intensity in the PCR amplification process is stronger, and the CT value is smaller.

It should be noted that, in order to remove the inhibitor inhibiting the PCR amplification reaction more cleanly, step (2) may be repeated for a plurality of times, such as 2 times, 3 times, or 4 times, and the specific times may be adjusted according to specific situations; the number of times of freezing and thawing can be increased, such as 3 times, 5 times or 8 times, and the specific times can be adjusted according to specific situations. In certain embodiments, the pre-processing method may comprise: (1) centrifuging milk to be detected in a 50mL centrifuge tube at 12000rpm for 5min, removing supernatant and fat, and inverting the centrifuge tube on a sterile filter paper sheet to remove the milk completely; (2) carrying out isometric resuspension by using sterile ultrapure water, centrifuging at 12000rpm for 5min, and retaining supernatant; (3) repeating the step (2) for 3 times; (4) performing metal bath on all the supernate obtained in the steps (2) and (3) at 100 ℃ for 6min, quickly freezing for 2min by using liquid nitrogen, and freezing and thawing for 5 times; taking a supernatant sample after vortex, and carrying out PCR amplification detection; it is to be noted that

It should be noted that the pretreatment of the cow milk to be tested is also a process of separating microorganisms from cow milk and extracting DNA, and it should be understood that other methods known in the art can be used in addition to the method for pretreatment of cow milk to be tested provided by the present invention.

In the method for detecting PCR amplification of bacillus thermotolerans, in some embodiments, the procedure of PCR amplification may be: 93 ℃ for 2 min; 93 ℃ for 10s, 56 ℃ for 30s, 40 cycles; the reaction system for PCR amplification can be: in each 20. mu.L reaction system, 0.8. mu.L of each of the upstream and downstream primers of the PCR amplification primer pair,

universal BlueqPCYBRGreenMasterMix 10 uL, DNA template to be detected 1 uL, ddH ₂ And O is supplemented to 20 mu L.

For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

In the following examples, q-PCR (real-time fluorescent PCR) reaction system (20. mu.L) is shown in Table 1 below,

TABLE 1 q-PCR reaction System (20. mu.L)

In the following examples, both BHI brain-heart extract medium and LB broth medium were obtained from Beijing Luqiao technology GmbH.

Example 1 isolation and identification of B.thermotolerans in cow's milk

(1) Preliminary screening of heat-resistant bacillus: heating a milk sample at 100 ℃ for 30min, pouring the milk sample into a BHI brain heart extract culture medium, culturing the milk sample at 20 ℃, 37 ℃ and 55 ℃ for 48-72 h, selecting a single colony, performing purification culture, and storing the strain subjected to purification culture in a freezing refrigerator at-80 ℃;

(2) strain activation and identification: inoculating the sterilized liquid stored in the freezing refrigerator at-80 ℃ in the step (1) into a BHI brain heart extract culture medium, culturing at 37 ℃ for 24-48 h, and continuously activating for 3 times until no mixed bacteria exist in microscopic examination (a microscopic examination picture is shown in figure 1); inoculating 2% of inoculum size into a BHI brain-heart extract culture medium, and taking a bacterial liquid to be sent to the Shanghai biological engineering for sequencing;

(3) separating and identifying heat-resistant bacillus in cow milk: sequencing the bacterial liquid in the step (2), and performing Blast in NCBI to find that the strain is highly homologous with the heat-resistant bacillus M215, wherein a phylogenetic tree is shown in figure 2, wherein J019, J020 and J021 are three repeated strain samples of the strain, and the homology of a 16srDNA sequence obtained by analysis and the M215 reaches 100%, which indicates that the strain belongs to the heat-resistant bacillus.

Example 2 design and validation of fluorescent quantitative PCR primers

(1) Test materials: listeria monocytogenes ATCC19115, Escherichia coli ATCC8739, Bacillus cereus CMCC (B)63303, Bacillus subtilis ATCC6633 and Citrobacter freundii ATCC43864 were all purchased from China Industrial microbial cultures Collection center (CICC), Bacillus thermotolerant M215(DSMZ10599) was purchased from German collection of microorganisms (DSMZ), and Bacillus thermotolerant B1 was purchased from cow's milk;

(2) activation of the strain: inoculating the above Bacillus cereus CMCC (B)63303, Bacillus subtilis ATCC6633, Listeria monocytogenes ATCC19115, Escherichia coli ATCC8739 and Citrobacter freundii ATCC43864 in LB broth, culturing at 37 ℃ to logarithmic phase for use; inoculating heat-resistant bacillus B1 and heat-resistant bacillus M215(DSMZ10599) in BHI brain heart leachate liquid culture medium, and culturing at 37 deg.C to logarithmic phase;

(3) designing a primer: comparing a 16srDNA sequence of the heat-resistant bacillus with bacillus, finding a highly conserved region of the bacillus, determining a V1-V9 variable region, designing a plurality of pairs of primers (shown in the following table 2) in the variable region through PrimerPremier6, verifying the specificity of the primers through calculating a Tm value and NCBI, and finally sending the designed primer sequence to Shanghai biological engineering for synthesis;

primer pairs designed in Table 2

(4) And (3) verifying the primers: detecting the specificity of the synthesized primers by using q-PCR, wherein a q-PCR reaction system is shown in the table 1 above, and a PCR reaction program is shown in the table 3 below;

TABLE 3 PCR PROGRAM (Procedure) in example 2

1	93.0℃for2:00
		2	93.0℃for0:10
3	60.0℃for0:30+PlateRead
		4	GOTO2,39moretimes
5	MeltCurve65.0to95.0℃,increment0.5℃,0:05+PlateRead
		END

The q-PCR results are shown in FIGS. 3 and 4, and the results were found by specific detection of a plurality of pairs of different primers (Table 2 above):

primer 1F1R (the sequence of 1F is shown as SEQ ID NO: 1, and the sequence of 1R is shown as SEQ ID NO: 2) has strong specificity, and in 7 different bacteria (Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Escherichia coli, citric acid freundii, M215 and Bacillus thermotolerant B1), only the B1 and M215 of the thermotolerant bacillus are subjected to specific starting, but the B bacillus subtilis and the Bacillus cereus are not started, the rest bacteria are not started, the CT value of fluorescence quantification is about 15, and the sensitivity is high (shown as figure 3);

while some of the remaining pairs of primers (2F2R, 3F3R, 4F4R, 5F5R and 6F6R) did not line bacillus thermotolerans B1 and M215, some did not line bacillus thermotolerans B1 and M215, and others such as bacillus subtilis and bacillus cereus, indicating that the remaining pairs of primers were not specific (as shown in fig. 4).

Example 3 Effect of different annealing temperatures on Heat-resistant Bacillus B1 in liquid Medium

In the embodiment of the present invention, a series of control reactions were set to determine the optimum annealing temperature, and 8 temperature gradients were set from 55 ℃ to 70 ℃ with the 8 temperature gradients being 55.0 ℃, 56.0 ℃, 58.0 ℃, 60.0 ℃, 64.5 ℃, 67.5 ℃, 69.2 ℃ and 70.0 ℃ respectively, and ddH as the control ₂ O; the q-PCR reaction system is shown in the above table 1, and the PCR reaction procedure is shown in the following table 4; the q-PCR assay was performed separately.

TABLE 4 PCR reaction procedure in example 3

1	93.0℃for2:00
		2	93.0℃for0:10
3	Gradient55.0/70℃for0:30，+PlateRead
		4	GOTO2,30moretimes
5	MeltCurve65.0to95.0℃,increment0.5℃,0:05+PlateRead
		END

The q-PCR detection result is shown in FIG. 5, and by setting annealing temperatures with different temperature gradients, the fluorescence intensity shows a gradually decreasing trend along with the temperature increase, and the CT value shows a gradually increasing trend; the higher the annealing temperature is, the greater the influence on the detection sensitivity of the heat-resistant bacillus B1 is; the detection sensitivity was highest at an annealing temperature of 60 ℃, so 60 ℃ was selected as the optimum annealing temperature for the subsequent experimental conditions.

Example 4 determination of detection Limit and Standard Curve for B1 of Bacillus thermotolerans in liquid culture Medium by primer 1F1R

In the embodiment of the invention, heat-resistant bacillus B1 is inoculated into a liquid culture medium BHI and cultured at 37 ℃ until logarithmic phase for later use; centrifuging heat-resistant Bacillus B1 cultured to logarithmic phase, resuspending with ultrapure sterilized water, resuspending, and adding ultrapure sterilized waterTen-fold gradient dilution, 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 Counting the bacteria liquid after gradient dilution by using a plate coating method, performing a q-PCR experiment on the rest bacteria liquid, extracting DNA of the undiluted heat-resistant bacillus B1 bacteria liquid to be used as a subsequent experiment for later use, and obtaining ddH ₂ O is a comparison; the q-PCR reaction system is shown in the above table 1, and the PCR reaction procedure is shown in the following table 5; q-PCR detection was performed separately.

TABLE 5 PCR reaction procedure in example 4

1	93.0℃for2:00
		2	93.0℃for0:10
3	60.0℃for0:30，+PlateRead
		4	GOTO2,39moretimes
5	MeltCurve65.0to95.0℃,increment0.5℃,0:05+PlateRead
		END

The q-PCR detection result showed that the primer q was found to be determined by measuring the detection limit of Bacillus thermotolerant B1 and the standard curveThe CT values of PCR are 15.48, 18.75, 21.74, 25.03, 28.15, 31.21, 34.82 and 36.68, and the concentrations of the corresponding heat-resistant bacillus B1 bacterial suspension are stock solution and 10 respectively ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 (ii) a Therefore, the primer 1F1R has high detection sensitivity and strong specificity on the heat-resistant bacillus B1; after counting by a plate, the bacterial fluid concentration of the heat-resistant bacillus which is not diluted is found to be 1.13 x10 ⁷ CFU/mL, combined with the spread plate method, the detection limit of primer 1F1R to B1 of Bacillus thermotolerans in liquid medium can reach 1CFU/mL, which indicates that the sensitivity is very high, as shown in FIG. 6; and the standard curve is shown in fig. 7, wherein the standard curve is that y is-3.0986 x +37.492, R ² 0.9979, indicating that the linearity is very high and the accuracy is very high.

Example 5 Effect of different cow milk treatment modes on the quantitative fluorescent detection of B1-Thermobacillus thermotolerans in cow milk

In the present example, the DNA of bacillus thermotolerant B1 was obtained from example 4, and the following different milk treatment methods were set up:

centrifuging 20mL of cow milk containing heat-resistant bacillus B1DNA in a 50mL centrifuge tube at 12000rpm for 5min, removing supernatant and fat, inverting the centrifuge tube on a sterile filter paper sheet to remove cow milk, and carrying out isometric resuspension on sterile ultrapure water.

And (2) centrifuging 20mL of cow milk containing the heat-resistant bacillus B1DNA in a 50mL centrifuge tube at 12000rpm for 5min, removing supernatant and fat, inverting the centrifuge tube on a sterile filter paper sheet to remove the cow milk, carrying out isometric resuspension on the sterile ultrapure water, centrifuging at 12000rpm for 5min, and retaining the supernatant, wherein the step is repeated for 3 times.

Thirdly, centrifuging 20mL of cow milk containing heat-resistant bacillus B1DNA in a 50mL centrifuge tube at 12000rpm for 5min, removing supernatant and fat, inverting the centrifuge tube on a sterile filter paper sheet to remove the cow milk completely, carrying out isometric resuspension on sterile ultrapure water, centrifuging at 12000rpm for 5min, retaining the supernatant, repeating the step for 3 times, then carrying out isometric resuspension on the sterile ultrapure water, and carrying out metal bath at 100 ℃ for 6min after resuspension.

And fourthly, centrifuging 20mL of cow milk containing the heat-resistant bacillus B1DNA in a 50mL centrifuge tube at 12000rpm for 5min, removing supernatant and fat, inverting the centrifuge tube on a sterile filter paper sheet to remove the cow milk completely, carrying out isometric resuspension on the sterile ultrapure water, centrifuging at 12000rpm for 5min, retaining the supernatant, repeating the step for 3 times, then carrying out isometric resuspension on the sterile ultrapure water, carrying out 6min in a metal bath at 100 ℃ after resuspension, quickly freezing for 2min by using liquid nitrogen, and freezing and thawing for 3 times.

Fifthly, centrifuging 20mL of cow milk containing heat-resistant bacillus B1DNA in a 50mL centrifuge tube at 12000rpm for 5min, removing supernatant and fat, inverting the centrifuge tube on a sterile filter paper sheet to remove the cow milk completely, carrying out isometric resuspension on sterile ultrapure water, centrifuging at 12000rpm for 5min, retaining the supernatant, repeating the step for 3 times, then carrying out isometric resuspension on the sterile ultrapure water, carrying out 6min in a metal bath at 100 ℃ after resuspension, quickly freezing for 2min by using liquid nitrogen, and freezing and thawing for 5 times.

And sixthly, centrifuging 20mL of cow milk containing the heat-resistant bacillus B1DNA in a 50mL centrifuge tube at 12000rpm for 5min, removing supernatant and fat, inverting the centrifuge tube on a sterile filter paper sheet to remove the cow milk completely, carrying out isometric resuspension on the sterile ultrapure water, centrifuging at 12000rpm for 5min, retaining the supernatant, repeating the step for 3 times, then carrying out isometric resuspension on the sterile ultrapure water, carrying out resuspension at 100 ℃ in a metal bath for 6min, quickly freezing for 2min by using liquid nitrogen, and freezing and thawing for 10 times.

Taking the cow milk treated in different modes, taking 1 mu L of the cow milk as a template loading amount to perform q-PCR, performing each experiment for 3 times, and performing ddH ₂ O as a control; the q-PCR reaction system is shown in Table 1 above, and the PCR reaction procedure is shown in Table 6 below; respectively carrying out q-PCR detection;

TABLE 6 PCR reaction procedure in example 5

1	93.0℃for2:00
		2	93.0℃for0:10
3	Gradient55.0/70℃for0:30，+PlateRead
		4	GOTO2,30moretimes
5	MeltCurve60.0℃+PlateRead
		END

The q-PCR results are shown in FIG. 8, and milk treated by different treatment methods will have a certain effect on the specific detection of Bacillus thermotolerant B1 by primer 1F1R, wherein the fifth treatment method can make the fluorescence stronger and the CT value smaller, so the fifth treatment method can be preferably used for pretreatment of milk.

Example 6 Effect of different milk sample additions on the quantitative fluorescent detection of B1 thermotolerant in cow's milk

In the present invention, the DNA of Bacillus thermotolerant B1 was obtained from example 4, and the milk pretreatment was performed using the fifth procedure, where the template loading was 0.1. mu.L, 0.2. mu.L, 0.5. mu.L, 1. mu.L, 2. mu.L, 5. mu.L, and ddH ₂ O is a comparison; the q-PCR reaction system is shown in Table 1 above (wherein, the amount of DNA template and ddH ₂ O adaptation), PCR reaction procedure is shown in table 7 below; respectively carrying out q-PCR detection;

TABLE 7 PCR reaction procedure in example 6

The q-PCR detection result is shown in FIG. 9, where different amounts of milk added have a certain influence on both the fluorescence intensity and the CT value, and the fluorescence intensity is the strongest and the CT value is the smallest when the template loading amount is 1. mu.L, so 1. mu.L can be preferably used as the template loading amount.

Example 7 Effect of different annealing temperatures on the quantitative fluorescent detection of B1, a heat-resistant bacterium in cow's milk

In the embodiment of the invention, different annealing temperatures are set, 8 temperature gradients are set from 55 ℃ to 70 ℃, the 8 temperature gradients are respectively 55.0 ℃, 56.0 ℃, 58.0 ℃, 60.9 ℃, 64.5 ℃, 67.5 ℃, 69.2 ℃ and 70.0 ℃, and the contrast is ddH ₂ O; the DNA of B1 was obtained from example 4, the q-PCR reaction system is shown in Table 1 above, and the PCR reaction procedure is shown in Table 8 below; q-PCR detection was performed.

TABLE 8 PCR reaction procedure in example 7

The q-PCR detection result is shown in FIG. 10, and the fluorescence intensity is strongest and the CT value is minimum when the annealing temperature is 56 ℃; it should be noted that the annealing temperature is optimized to some extent because Bacillus thermotolerans B1 detected in cow's milk and Bacillus thermotolerans B1 detected in liquid medium are different (cow's milk contains substances that may inhibit PCR reaction, such as fats).

Example 8 determination of detection Limit and Standard Curve for B1B Heat-resistant Bacillus Calmette in cow's milk by primer 1F1R

In the embodiment of the invention, the DNA of the heat-resistant bacillus B1 is obtained in the embodiment 4, and the pretreatment mode of the milk is selected as a fifth treatment mode; it was then diluted in a ten-fold gradient, 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Samples after gradient dilution, in ddH ₂ O is a comparison; the q-PCR reaction system is shown in Table 1 above, and the PCR reaction procedure is shown in Table 9 below; carrying out q-PCR detection;

TABLE 9 PCR reaction procedure in example 8

1	93.0℃for2:00
		2	93.0℃for0:10
3	56.0℃for0:30，+PlateRead
		4	GOTO2,39moretimes
5	MeltCurve65.0to95.0℃,increment0.5℃,0:05+PlateRead
		END

The q-PCR detection result shows that the CT values of the primer q-PCR are 21.7, 25.35, 29.14, 32.76, 35.9 and 39.06 through the detection limit of the heat-resistant bacillus B1 in the milk and the measurement of a standard curve, and the concentrations of the corresponding heat-resistant bacillus B1 bacterial suspensions are respectively stock solution and 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 (ii) a Incorporating the Heat-resistant spores of example 4The viable count corresponding to the DNA of the bacillus B1 shows that the detection limit of the primer 1F1R on the heat-resistant bacillus B1 in the milk can reach 11CFU/mL, which indicates that the sensitivity is very high, as shown in FIG. 11; the standard curve is shown in fig. 12, and is y-3.4877 x +43.044, R ² 0.9984, indicating that the linearity is very high and the accuracy is also very high.

Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which shall be covered by the claims of the present invention.

Sequence listing

<110> Tianyou Dairy Co Ltd of Chongqing City

<120> PCR amplification primer pair and application thereof

<130> 2022-6-16

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tggtacaaag ggttgctaga 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggttttatgg gattggcgta 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caaggctgaa actcaaagga 20

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tctagggagg tcaagaggat g 21

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tctgtaactg acgctgaggc 20

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agcactaaag ggcggaaa 18

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caaggctgaa actcaaagga 20

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgaatgctgg caactaaggt c 21

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acaagagtga caggtggtgc 20

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcctacaatc cgaactgaga a 21

<210> 11

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acggctcaac cgag 14

<210> 12

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtaacctcgc ggtct 15

Claims

A PCR amplification primer pair, wherein the upstream primer comprises the sequence set forth in SEQ ID NO: 1, and the downstream primer comprises a sequence shown as SEQ ID NO: 2.
2. A detection reagent comprising the PCR amplification primer set according to claim 1.
3. The detection reagent according to claim 2, further comprising a labeling reagent, wherein the labeling reagent includes but is not limited to a fluorescent dye or a fluorescent probe.
4. A detection kit comprising the PCR amplification primer set according to claim 1 or the detection reagent according to any one of claims 2 to 3.
5. Use of the PCR amplification primer pair of claim 1 or the detection reagent of any one of claims 2 to 3 or the detection kit of claim 4 for detecting bacillus thermotolerans.
6. The PCR amplification detection method of the heat-resistant bacillus is characterized by comprising the following steps: carrying out PCR amplification on a DNA template to be detected by using the PCR amplification primer pair of claim 1 to obtain a PCR amplification product, and analyzing the PCR amplification product so as to judge whether the DNA template to be detected contains heat-resistant bacillus.
7. The PCR amplification detection method of the heat-resistant bacillus is characterized by comprising the following steps: mixing a DNA template to be detected with the detection reagent according to claim 3, and then carrying out PCR amplification, wherein in the PCR amplification process, whether heat-resistant bacillus exists in the DNA template to be detected or not is judged according to a fluorescent signal; and judging the content of the heat-resistant bacillus in the DNA template to be detected according to the CT value and the standard curve.
8. The method for PCR detection of Bacillus thermotolerant according to claim 6 or 7, wherein the annealing temperature for PCR amplification is 56 ℃ to 60 ℃.
9. The method for PCR detection of Bacillus thermotolerans according to claim 6 or 7, wherein the concentrations of the DNA template to be detected are: the concentration of the DNA template to be detected is less than 2 mu L/20 mu L and is less than 0.5 mu L/20 mu L.
10. The method for detecting the PCR amplification of Bacillus thermotolerant according to claim 6 or 7, wherein the Bacillus thermotolerant is Bacillus thermotolerant in cow's milk, and the method for detecting the PCR amplification of Bacillus thermotolerant further comprises a pretreatment of the cow's milk to be detected before the PCR amplification detection, wherein the pretreatment comprises: (1) centrifuging the milk to be detected to remove supernatant and fat, and filtering to remove the milk completely; (2) carrying out isometric resuspension by using sterile pure water, and centrifuging to retain supernatant; (3) the supernatant is subjected to metal bath at 95-105 ℃, quick-frozen and freeze-thawed.