CN111304301A

CN111304301A - Application of BAX PCR method in detection of salmonella in food

Info

Publication number: CN111304301A
Application number: CN202010111452.2A
Authority: CN
Inventors: 贺梦醒
Original assignee: Eurofins Technology Service Suzhou Co ltd
Current assignee: Eurofins Technology Service Suzhou Co ltd
Priority date: 2020-02-24
Filing date: 2020-02-24
Publication date: 2020-06-19

Abstract

The invention relates to an application of a BAX PCR method in food salmonella detection; step one, placing a sample in enrichment broth to prepare initial suspension; step two, adding the enrichment fluid extracted in the step one into the brain-heart infusion broth, and culturing for the second enrichment; step three, adding the enrichment liquid re-cultured by the second enrichment in the step two into a cracking tube, adding the cracking liquid containing the protease into the cracking tube, heating the cracking tube in a heating tube, cooling the cracking tube in a cooling block, and then taking the cracking product to be placed in a PCR reaction tube; step four, performing on-machine detection on the PCR reaction tube in the step three; the invention provides an application of a BAX PCR method in food salmonella detection, which is applied to the salmonella detection in food, thereby rapidly carrying out the salmonella detection of a large batch of samples.

Description

Application of BAX PCR method in detection of salmonella in food

Technical Field

The invention relates to the technical field of food detection, in particular to application of a BAX PCR method in food salmonella detection.

Background

Salmonella is a group of gram-negative, facultative anaerobes that colonize the intestinal tracts of humans and animals. The antigenic types of the genus are very complex, and it is reported that there are over 2500 serotypes worldwide, and there are over 220 serotypes in our country. As a common food-borne pathogenic bacterium, the condition that the common food-borne pathogenic bacterium pollutes most animal-borne foods such as eggs, livestock and poultry meat, fresh milk and the like is very common, and the common food-borne pathogenic bacterium also widely exists in natural environment and is the primary pathogenic bacterium for causing food-borne bacterial diseases. In Europe, hundreds of thousands of people are infected with salmonella every year, in the United states, hundreds of thousands of infected salmonella are infected with salmonella every year, and 70% -80% of bacterial food poisoning events in China are caused by salmonella, so that in the 80 th century as early as 20 th, the infection problem of salmonella causes high attention of experts in food fields at home and abroad, and corresponding monitoring limit standards are established, and are definitely given no matter the European Union standard or the national standard (GB29921-2013 food safety national standard for pathogenic bacteria limit): the salmonella detection adopts a three-level sampling method, and the salmonella cannot be detected under any condition.

The traditional salmonella detection method mainly adopts a selective culture medium and biochemical reaction characteristics for detection, has long period, complicated process and low sensitivity, and is difficult to meet the rapid requirement of food sanitation detection. The following drawbacks exist: 1) the traditional method requires at least 6 isolation culture media, nearly 10 identification reagents and expensive identification strips; 2) the complexity of the types of the salmonella antigens, the diversity of biochemical characteristics and the judgment difficulty of the identification result are high, and the control can be well realized by experimenters with rich experience; 3) the variety of food matrixes is various, the influence of matrix inhibition is infinite, and the sensitivity of the traditional method is difficult to meet; 4) the novel selective chromogenic culture media contain high-strength antibacterial substances, and have certain harm to human bodies after long-term contact; 5) food safety is increasingly emphasized, detection requirements are continuously strengthened, the requirements for large-batch samples and large-volume sampling amount are more and more, and the traditional method is difficult to efficiently meet.

In recent years, researchers devote to the development of new salmonella detection technologies, and make great progress from a time-consuming and labor-consuming traditional separation culture method to an immunological detection method and then to a molecular biological detection technology, and the application aims to provide the application of a BAX PCR method with high detection efficiency in the detection of salmonella in food.

Disclosure of Invention

The invention aims to provide a rapid real-time fluorescence quantitative PCR detection technology, which is applied to the detection of salmonella in food, so that the BAX PCR method for rapidly detecting salmonella in a large batch of samples is applied to the detection of the salmonella in food.

In order to achieve the above object, the present invention provides an application of a BAX PCR method in salmonella food detection, comprising the following steps:

step one, placing a sample in enrichment broth to prepare initial suspension;

step two, adding the enrichment fluid extracted in the step one into the brain-heart infusion broth, and culturing for the second enrichment;

step three, adding the enrichment liquid re-cultured by the second enrichment in the step two into a cracking tube, adding the cracking liquid containing the protease into the cracking tube, heating the cracking tube in a heating tube, cooling the cracking tube in a cooling block, and then taking the cracking product to be placed in a PCR reaction tube;

and step four, performing on-machine detection on the PCR reaction tube in the step three.

Further, the enrichment fluid in the first step comprises one or more of BPW/lactose broth/tryptone soy broth.

Further, the second enrichment re-culture in the second step is a culture at 37 ℃ for 3 hours.

Further, the cracking tube in the third step is heated in heating plates at 37 ℃ and 95 ℃ in sequence.

Further, the pyrolysis tube in step three was heated in a heating plate at 37 ℃ for 20 minutes.

Further, the lysis tube in step three was placed in a heating plate at 95 ℃ for 10 minutes.

Further, the cracking tube in the third step was cooled in a cooling block for 5 minutes.

According to the technical scheme of the invention, the application has the following advantages:

1) the time for the negative result of the salmonella is shortened from 4 days to 2 days, and the time for the positive result of the salmonella is shortened from 7 days to 4 days;

2) the method is simple to operate, is more convenient than the conventional PCR method, does not need to design and prepare a PCR reaction system, and saves more manpower, material resources and financial resources;

3) the method is approved by multiple international standards, the sensitivity and specificity of the method are strongly guaranteed, and the detection result is more accurate.

The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention.

Drawings

FIG. 1 is a peak value of a strong positive sample of the present invention;

FIG. 2 is a peak value of a medium positive sample of the present invention;

FIG. 3 is a peak value of a strong positive sample of the present invention;

FIG. 4 is a peak value of a weakly positive sample of the present invention;

FIG. 5 is a peak value of a negative sample of the present invention.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Examples

step one, placing a sample in enrichment broth to prepare initial suspension;

In this example, the enrichment medium in step one is typically BPW, and further lactose broth and trypticase soy broth, etc., and the enrichment medium selected differs depending on the substrate.

In this example, the selective enrichment culture in the second step was carried out at 37 ℃ for 3 hours.

In this example, the pyrolysis tube in step three was heated in heating plates at 37 ℃ and 95 ℃ in this order.

In this example, the lysis tubes in step three were heated in a 37 ℃ hot plate for 20 minutes.

In this example, the lysis tubes in step three were placed in a heating plate at 95 ℃ for 10 minutes.

In this example, the cracking tube in step three was cooled in a cooling block for 5 minutes.

Embodiment 1

1) Sample treatment: weighing 1g of sample into 9ml of pre-enrichment solution BPW to prepare 1:10 initial suspension;

2) selective enrichment: after enrichment in the step 1), adding 10 mul of the pre-enrichment liquid into 0.5ml of brain-heart infusion broth, and culturing for 3 hours at 37 ℃;

3) cracking: after the enrichment is finished, a PCR detector is started to preheat, heating plates at 37 ℃ and 95 ℃ are started simultaneously, 150 mu L of protease is added into 12ml of lysis solution, 200 mu L of lysis solution is taken into each lysis tube participating in the reaction, and then 5 mu L of enrichment solution is added. The lysis tube was placed on a 37 ℃ hot plate for 20 minutes, after which the tube was moved to a 95 ℃ hot plate for 10 minutes, the tube was cooled in a cooling block for 5 minutes, after which 50. mu.L of lysate was carefully removed to the PCR reaction tube placed in the cooling block (handling should avoid contamination);

4) and (3) loading: placing the PCR tube covered with the tube cover in a PCR detector, and selecting a salmonella program for detection;

5) and (3) reading a result: after PCR amplification, the results of the experiment will be displayed directly on a computer. A "-" symbol indicates a negative result, a "+" symbol indicates a positive result, "? The "symbol indicates an indeterminate result, and the question mark plus the slash symbol indicates a signal error. Identification is required except for negative results.

6) And (3) data analysis:

positive dissolution curve

a. The three target peaks are located at: 85 ℃, 88 ℃ and 90 ℃;

b. the target peak range is: 82-91 ℃;

c. the internal reference quality control peak value range is as follows: 76-80 ℃.

When the sample is strongly positive, peaks at 88 ℃ and 90 ℃ appear and are obvious, in this case, the reference control peak appears to be small or even no, and four positive result peak cases are shown in fig. 1 to 4.

Negative dissolution curve

No target peak value exists, and only an internal reference peak value appears; in some cases, a small nonspecific peak appears in the 84-92 ℃ region, and the positive result cannot be judged, as shown in FIG. 5.

In summary, the following steps:

1) the method has been applied for multiple times to participate in international performance verification (Fapas238, LGC QMS MC268, LGCQMS229, LGC AF039 and LGC QCS 227) and achieve satisfactory results;

2) the method has the advantages that the false negative is easily caused in the traditional method for the low-pollution sample, the sensitivity of the method can reach 104cfu/ml, and the false negative condition is greatly reduced;

3) in daily detection, some samples which are not always positive are rechecked by the technology, so that the accuracy of the result is ensured.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. The application of a BAX PCR method in the detection of salmonella in food is characterized by comprising the following steps:

step one, placing a sample in enrichment broth to prepare initial suspension;

2. The method of claim 1, wherein the step of: the enrichment fluid in the first step comprises one or more of BPW/lactose broth/trypticase soy broth.

3. The method of claim 1, wherein the step of: the second enrichment and re-culture in the second step is to culture for 3 hours at 37 ℃.

4. The method of claim 1, wherein the step of: the cracking tube in the third step is heated in heating plates at 37 ℃ and 95 ℃ in sequence.

5. The method of claim 4, wherein the step of: the lysis tubes in step three were heated in a heating plate at 37 ℃ for 20 minutes.

6. The method of claim 4, wherein the step of: the lysis tubes in step three were placed in a heating plate at 95 ℃ for 10 minutes.

7. The method of claim 1, wherein the step of: the cracking tube in step three was cooled in a cooling block for 5 minutes.