CN101045943A - Process of PCR detecting salmonella with added amplifying internal standard - Google Patents

Abstract

The invention relates to a method for detecting Salmonella by PCR with an amplified internal standard in the technical field of food safety. The specific steps of the present invention are as follows: (1) Design specific primers. (2) By optimizing the concentration of MgCl ₂ and the value of annealing temperature Tm, the PCR reaction system is in the optimal reaction condition. (3) Select the concentration of the amplification internal standard that has the least impact on the detection sensitivity and can clearly indicate false negatives as the added concentration. (4) Perform PCR detection to determine whether the specificity of the primers and the amplification internal standard can be amplified normally in the detection of non-Salmonella samples. (5) The established PCR reaction system detects artificially polluted samples and naturally polluted samples. The invention helps the PCR detection system to be able to show the occurrence of false negatives, avoids misjudgment of results due to false negative detection results caused by inhibitors in samples or operational errors, thereby improving the accuracy of PCR detection.

Description

A method for detection of Salmonella by PCR with amplified internal standard

technical field

The invention relates to a detection method in the technical field of food safety, in particular to a detection method for Salmonella PCR with an amplified internal standard added.

Background technique

PCR, the abbreviation of polymerase chain reaction in English, refers to: polymerase chain reaction. The establishment of an accurate and rapid PCR detection method for foodborne pathogens can promote the timely and effective prevention, early warning and control of foodborne diseases in my country and protect the health of the people. After China's accession to WTO, food safety issues have become the main obstacle restricting my country's foreign trade, seriously hindering economic development and affecting people's health. Therefore, it is a national demand to solve food safety problems through the development of rapid detection technology. my country's current national standard inspection method for Salmonella, an important pathogenic bacteria in food, adopts the traditional culture method. Because this method is time-consuming and laborious, it can no longer meet the needs of rapid detection of pathogenic bacteria in food processing and production.

In 1992, Rahn et al. took the lead in designing a pair of primers based on the invA gene of Salmonella typhimurium for the PCR detection of Salmonella. For details, see: Rahn K, De grandis S A, Clarke R C, et al.Amplification of an invA gene sequence of Salmonella typhimurium bypolymerase chain reaction as a specific method of detection of Salmonella. Molecular and Cellular Probes.1992, 6(4): 271-279. (Rahn K, De grandis S A, Clarke R C, etc. with typhoid fever The invA gene sequence of Salmonella is used as a PCR detection gene for the detection of Salmonella. Molecular and Cellular Probes. 1992, 6(4): 271-279.). Since then, PCR detection technology for Salmonella has been widely used, and the number of genes used for detection is increasing, and the primers designed are also increasingly diverse, but most researchers still use Rahn's detection primers. The detection of Salmonella by PCR detection technology has the characteristics of rapidity, sensitivity and specificity. However, most of the detection primers are designed before the development of gene sequencing and bioinformatics, and there are occasional false detections and missed detections, resulting in false positive and false negative results. However, there are certain differences in the target genes and operating procedures detected by PCR methods in different laboratories or testing departments. There is no standard, and the test results obtained are not the same. Practical applications in recent years have shown that inhibitors in food and culture media can affect PCR reactions and make detection results false negative. Although the PCR method is continuously improved and perfected, it cannot effectively prevent the occurrence of false negative results.

Some scholars have begun to realize that it is necessary to place an internal amplification control (IAC) indicating false negatives in the PCR reaction system. In 2003, Malorny et al. used the method of molecular cloning to artificially construct a 157bp amplified internal standard fragment using a partial fragment of the invA gene, and could indicate the occurrence of false negatives in the test results. See: Malomy B, Hoorfar J, Bunge C, et al. Multicenter Validation of the analytical accuracy of Salmonella PCR: toward an international standard. Applied and environment microbiology. 2003, 69(1): 290-296. (Malomy B, Hoorfar J , Bunge C, et al. Multi-channel confirmation of the correctness of Salmonella PCR detection: Expectation of an international standard. Applied and Environmental Microbiology. 2003, 69(1): 290-296.). However, the amplified internal standard constructed by it has high homology with the detected invA gene, which will interfere with the hybridization of the detection primer and the detection gene to varying degrees during detection; the detection primer used is still Rahn in 1992. When the reported primers detect some Escherichia coli (E.coli), non-specific bands will appear, which reduces the specificity of the reaction system.

In view of the above problems, this study used the method of bioinformatics to redesign the detection primers based on the invA gene, and the construction of the amplification internal standard mainly came from another specific gene (stn gene) of Salmonella, which is different from the detection gene invA gene of Salmonella. Homologous and non-homologous to non-Salmonella genomes.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting Salmonella PCR with an internal standard for amplification. Adding the constructed amplified internal standard fragment to the PCR reaction system will help to show the occurrence of false negatives during detection, improve the accuracy rate, and meet the urgently needed investigation of food-borne pathogens in the process of quarantine law enforcement And detection provides effective technical means.

The present invention is realized through the following technical solutions, and the concrete steps of the present invention are as follows:

(1) Use the specific gene sequence of Salmonella to construct an internal standard for amplification and design specific primers.

(2) By optimizing the concentration of MgCl ₂ and the value of annealing temperature Tm, the PCR reaction system is in the optimal reaction condition. First, according to the Tm value recommended when designing the primers, set the temperature gradient at intervals of 1°C within the range of Tm±5°C, and select the annealing temperature according to the brightness of the amplified product in the electropherogram. Set a series of Mg ²⁺ concentration gradients, from 1.0mmol/L to 3.0mmol/L (interval 0.5mmol/L), and select the Mg ²⁺ of the PCR reaction system according to the brightness of the amplified product in the electropherogram. concentration.

(3) The determined total DNA solution of Salmonella was diluted 10-fold with sterile water to 10 ^-10 . Using the diluted DNA solution as a template, 5 μL was added to the PCR reaction system for PCR detection, and at the same time, it was replaced with sterile water. The DNA template is used as a negative control, and the detection sensitivity of PCR is selected. Then, the carrier with the amplified internal standard is taken and added to the PCR reaction system. Amplified internal standards of different dilution concentrations were added to the reaction system. Again, for the sensitivity of detecting DNA or bacterial strains, the concentration of the amplified internal standard that had the least impact on the detection sensitivity and could clearly indicate false negatives was selected as the added concentration.

(4) Extract the DNA of Salmonella standard strains of different serotypes and non-Salmonella standard strains of different species, and perform PCR detection to determine the specificity of primers and whether the amplification internal standard can be amplified normally in the detection of non-Salmonella samples. In the results, all Salmonella should be positive and all non-Salmonella should be negative.

(5) The established PCR reaction system detects artificially polluted samples and naturally polluted samples. By detecting artificially polluted samples, it is judged whether the amplification internal standard indicates a false negative; by detecting naturally polluted samples, the national standard method and the test results of API reagent strips are used for reference to evaluate whether the test results of the established PCR detection system are accurate.

Detection of artificially contaminated samples: collect 80 milk samples with serious artificial contamination, take 25mL of each sample, transfer to 225mL BPW liquid medium, enrich the bacteria in a shaker (150r/min) at 37°C, and increase the bacteria for 8h After sampling. Take 1 mL of each sample, put it into a 1.5 mL centrifuge tube, and centrifuge at 3 000 r/min for 10 min to precipitate food residues in the culture. Take the supernatant, centrifuge at 12 000r/min for 5min, and collect the Salmonella thallus. After pouring off the supernatant, resuspend the cells with sterile double distilled water, and centrifuge and wash three times. Then add 100 μL of sterile ultrapure water and cook in a boiling water bath for 10 min. Immediately after taking it out from the boiling water bath, place it at -20°C for 30min. After thawing at 37°C, centrifuge at 12 000r/min for 5min, and the supernatant was the PCR template DNA solution, and 5 μL was added to the PCR reaction system for PCR detection, and sterile water was used instead of the DNA template as a negative control.

Detection of naturally contaminated food samples: 50 eggs were collected, 25mL of each egg was transferred into 25mL sterilized normal saline, homogenized, and 25mL samples were homogenized into 100mL magnesium chloride malachite green enrichment solution (MM) and 100mL sub Nitrate cystine enrichment solution (SC) for selective enrichment culture. After the samples inserted into the nitrite cystine enrichment solution were incubated at 36°C for 8 hours, 1 mL was sampled from each sample for PCR detection. After 24 hours of selective enrichment culture, they were separated on bismuth sulfite (BS) agar plate and DHL agar plate, and suspicious colonies were collected from the two selective plates, and further detected and identified by biochemical tests and API reagent strips.

The amplified internal standard DNA sequence mainly comes from the stn gene of Salmonella, and the two ends of the DNA sequence respectively contain the sequences of detection primers. Its purpose is to show the occurrence of false negatives and improve the accuracy of PCR detection.

The construction of the internal standard for amplification refers to: the use of the specific gene sequence of Salmonella itself to construct the internal standard for amplification, which reduces the interference of the DNA of other organisms other than Salmonella on PCR detection, and also avoids the interference of the internal standard for amplification. Homologous cross-linking interference of the target gene. The amplified internal standard reduces the interference on the target gene during the detection process and increases the accuracy of detection.

The method for amplifying the internal standard is as follows: use sequence analysis and gene comparison to select the specific gene sequence used to construct the amplified internal standard for Salmonella PCR detection, and have extremely low homology with the target gene for detection; use gene cloning The two ends of the selected gene sequence were respectively connected with detection primers to construct an internal standard for amplification, and cloned into the vector; the above-mentioned vector with internal standard for amplification was transferred to Escherichia coli (E.coli 5α) by transgenic method Then spread it on a selective plate (90mm petri dish, 10μl of 100mg/ml ampicillin, 7μl of 20% IPTG solution, 40μl of 2% X-gal solution), and incubate at 37°C for 12h. Use a sterilized toothpick to pick the white colony from the selective plate, inoculate it into a test tube containing 5ml of LB culture solution, and then culture it at 150r/min at 37°C for 6h, extract the DNA of plasmid pMD-stn, and perform PCR After amplification, it was confirmed that transformants were obtained.

The host cell transformed by the vector, which is Escherichia coli in an example, contains amplified internal standard DNA fragments.

The specific primers refer to: a pair of detection primers (psL/R) for the PCR detection of Salmonella, which can be amplified to the specific detection gene of Salmonella strains during PCR detection, or added in the PCR system There are two pairs of amplification primers (sinL/R) and long primers (sivL/R) for constructing the amplification internal standard; the amplification primers (sinL/R) can only be amplified to the Amplify the DNA fragment of the internal standard, and the long primer (sivL/R) can obtain the internal standard for amplification by means of PCR amplification and gene cloning.

A section of the present invention is used to construct amplified internal standard DNA sequence, which is from the specific gene stn gene of Salmonella.

The target gene refers to the invA gene of Salmonella.

The false negative refers to that the PCR reaction is affected by the inhibitors in the food or the culture medium and no reaction occurs, or the result is negative due to the operation error of the operator.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. A gene sequence used as an internal standard for amplification is operably linked to an expression control sequence, thereby carrying an internal standard for amplification.

The PCR detection method of the present invention adds a fragment of an amplified internal standard on the basis of the common PCR method. In the same PCR amplification process, the same detection primer can be used to simultaneously amplify the target gene. When the DNA content of Salmonella in the sample is higher than the detection sensitivity, the electrophoresis result contains the amplified fragment of the target gene, and the detection result is positive; when the sample does not contain or is lower than the detection sensitivity, the electrophoresis result only contains the amplified fragment. The target amplified fragment, the test result is negative; when the electrophoresis result does not contain any amplified fragment, the test result is a false negative. The method helps the PCR detection system to display the occurrence of false negatives, and avoids misjudgment of results due to false negative detection results caused by inhibitors in the sample or operational errors, thereby improving the accuracy of PCR detection.

Description of drawings

Figure 1 Schematic diagram of the sensitivity of Salmonella choleraesuis detection without adding IAC

In the figure: electrophoresis lanes 1 to 8: the template DNA concentrations contained in each PCR reaction are 12.8ng/μL, 1.28ng/μL, 128pg/μL, 12.8pg/μL, 1.28pg/μL, 128fg/μL, 12.8fg/μL, 1.28fg/μL. M: 100bp DNA molecular weight standard.

Figure 2 Schematic diagram of the sensitivity of Salmonella choleraesuis detection when adding IAC to 3.65× ¹⁰⁴ copies

In the figure: electrophoresis lanes 1 to 8: the template DNA concentrations contained in each PCR reaction are 12.8ng/μL, 1.28ng/μL, 128pg/μL, 12.8pg/μL, 1.28pg/μL, 128fg/μL, 12.8 fg/μL, 1.28 fg/μL; electrophoresis lane 9: negative control (dH ₂ O). M: 100bp DNA molecular weight standard.

Figure 3 Schematic diagram of the detection of colony sensitivity

In the figure: electrophoresis lanes 1-10: the number of initial colonies of Salmonella choleraesuis added to each PCR reaction is 3.7×10 ⁷ , 3.7×10 ⁶ , 3.7×10 ⁵ , 3.7×10 ⁴ , 3.7×10 ³ , 3.7×10 ² , 37, 3-4, 0-1, 0 cfu/mL. M: 100bp DNA molecular weight standard.

Figure 4 Schematic diagram of anti-interference test

Detailed ways

Below in conjunction with specific embodiment, further elaborate the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example

1. Preliminary establishment of the detection system

1. Select the target gene for detection

The known specific genes of Salmonella were analyzed by bioinformatics, and the target gene invA was selected for detection. Using BLAST and other software, compare the sequence of the invA gene with other microorganisms, and select the sequence segment with higher specificity. Then use the software Primer 5.0 to design a pair of internal standard primers in this specific sequence. The primer sequences are as follows:

psL: 5'- TGTCACCGTGGTCCAGTTTA -3'

psR: 5'- CGACAAGACCATCACCAATG -3'

(a) Detect the sequence characteristics of the target gene:

^* Length: 364 bp

^* Type: nucleic acid

^* Chain type: double chain

^* Topology: linear

(b) Molecule type: DNA

(c) Original source: Salmonella strains

(d) Sequence description: SEQ ID NO.1:

TGTCACCGTGGTCCAGTTTA TCGTTATTACCAAAGGTTCAGAACGTGT

CGCGGAAGTCGCGGCCCGATTTTCTCTGGATGGTATGCCCGGTAAACAGAT

GAGTATTGATGCCGATTTGAAGGCCGGTATTATTGATGCGGATGCCGCGCGC

GAACGGCGAAGCGTACTGGAAAGGGAAAGCCAGCTTTACGGTTCCTTTGA

CGGTGCGATGAAGTTTATCAAAGGTGACGCTATTGCCGGCATCATTATTATC

TTTGTGAACTTTATTGGCGGTATTTCGGTGGGGATGACTCGCCATGGTATGG

ATTTGTCCTCCGCCCTGTCTACTTATACCATGCTGAC CATTGGTGATGGTCTT

GTCG

The underlined letters in the sequence represent the positions of primers psL and psR.

2. Optimization of PCR reaction system

Through the optimization of annealing temperature and Mg2 ⁺ concentration, the determined reaction system is as follows:

10×buffer 2.5μl

MgCl ₂ solution (25mM) 1.5μl

dNTP(2.5mM) 1.0μl

Upstream and downstream primers (10μM each) 1.0μl+1.0μl

Taq DNA polymerase (2.5U/μl) 0.3μl

Template 5.0μl

Add sterile water to 25μl

PCR cycle parameters are: pre-denaturation at 94°C for 3 min, followed by 35 cycles, each cycle includes denaturation at 94°C for 30 s, the annealing temperature is selected according to the following steps, the annealing time is 30 s, and then extended at 72°C for 30 s, cycle After the end, extend at 72°C for 10 minutes, and finally cool down to 12°C to end all operating procedures.

2. Amplification of the gene sequence used to prepare the internal standard for amplification

1. Select the gene used to prepare the internal standard for amplification

A specific DNA sequence of the stn gene of Salmonella was selected to construct an internal standard for amplification. The stn gene is one of the specific genes of Salmonella. Selecting the stn gene to prepare the amplified internal standard can not only ensure that the sequence of the amplified internal standard constructed has low homology with the detected target gene, but also has low homology with the total DNA sequence of other microorganisms. source is also low.

2. Select the specific segment of the stn gene and design primers

The sequences of Salmonella stn gene and invA gene were searched in GenBank. Using software such as BLAST, compare the sequence of the stn gene with other microorganisms, and select the sequence segment I with higher specificity. Then, the sequence segment I was compared with the sequence of the invA gene of Salmonella, and a DNA sequence segment II with the lowest homology was found. Then use the software Primer 5.0 to design a pair of internal standard primers in this sequence segment II. The primer sequences are as follows:

sinL: 5'-TGGGCATCCTGACTGACCAG-3'

sinR: 5'-CGACGAACCAGCGAAACAAA-3'

(a) Construct the gene sequence characteristics of the internal standard of amplification:

^* Length: 476 bp

^* Type: nucleic acid

^* Chain type: double chain

^* Topology: linear

(b) Molecule type: DNA

(c) Original source: Salmonella strains

(d) Sequence description: SEQ ID NO.2:

TTCAGGGAGTGAGTAATAAT

TTCAGGGAGTGAGTAATAAT

ATCATTGAGGTTAACCGTCTGGAGCGTCAGATGCGCGGGCTTTAC

CAGTTCGAGCAATTCGCTTACCACCCGGTTCAAACGGTCGGCCTC

TTTGGCCATCACCTGCGCCAGTTCATGCGACTCGCCGCCGGCAGG

CGTGCGCTCGGCAAAGTATTTCGCCAGCCCTTTGATGGACGAGAG

CGGGTTACGAATTTCGTGCGCGACGCCCGCCGCCAGATGCCCCAT

CGCCACCAGCTTTTCTTTACGCTTCATTGCATCAAGCAGTTCTCTG

TGCGAGCGCTGATAACGCTGATGCCAAAAAAACGCCAGTAACGT

TGCCAACAGCACCGCCGCTAACGCAGACAAAACAATCAGCGTAT

TATTTACTCCCTTGCCTGTGTCGCGGCCACGTCGCTGTCAAAGGC

AATAAAAATAG

The shaded letters in the sequence represent the positions of the primers sinL and sinR.

3. Connect

The amplified product fragments were recovered according to the instructions of the PCR Recovery Kit. The recovered amplified product fragment and the pMD18-T vector were incubated overnight at 16°C under the action of Ligation Solution I.

4. Transformation of E. coli

Escherichia coli was transformed by calcium chloride method, and positive clones of transformed E. coli were detected by blue-white screening.

5. Detection

Using specific primers (sinL and sinR) to perform PCR detection on the screened E. coli positive clones, and to further sequence and verify the DNA of the extracted plasmid pMD-stn from the E. coli positive clones determined by PCR detection.

3. Preparation of internal standard for amplification

1. Design long primers

The target primers psL and psR were respectively connected to the 5' end of the designed stn internal standard primer to form a pair of long primers of about 40 bp. The primer sequences are as follows:

-3’sivL: 5'- TGTCACCGTGGTCCAGTTTA

-3'

sivR :

-3’5'- CGACAAGACCATCACCAATG

-3'

2. PCR amplification to obtain the amplification internal standard

The DNA of plasmid pMD-stn was amplified with long primers sivL and sivR, and the amplified product was the internal standard of amplification.

(a) Sequence characteristics of the amplified internal standard:

^* Length: 516 bp

^* Type: nucleic acid

^* Chain type: double chain

^* Topology: linear

(b) Molecule type: DNA

(c) Original source: Salmonella strains

(d) Sequence description: SEQ ID NO.3:

AT TGTCACCGTGGTCCAGTTTA

AT

TCAGGGAGTGAGTAATAATATCATTGAGGTTAACCGTCTGGAGC

GTCAGATGCGCGGGCTTTACCAGTTCGAGCAATTCGCTTACCACC

CGGTTCAAACGGTCGGCCTCTTTGGCCATCACCTGCGCCAGTTCA

TGCGACTCGCCGCCGGCAGGCGTGCGCTCGGCAAAGTATTTCGCC

AGCCCTTTGATGGACGAGAGCGGGTTACGAATTTCGTGCGCGAC

GCCCGCCGCCAGATGCCCCATCGCCACCAGCTTTTCTTTACGCTT

CATTGCATCAAGCAGTTCTCTGTGCGAGCGCTGATAACGCTGATG

CCAAAAAAACGCCAGTAACGTTGCCAACAGCACCGCCGCTAACG

CAGACAAAAACAATCAGCGTATTATTTACTCCCTTGCCTGTGTCGC

GGCCACGTCGCTGTCAAAGGCAATAAAATAG

CGACAAGACCATCACCAATG

The underlined and shaded letters in the sequence represent the positions of primers sivL and sivR, respectively.

3. Connect

The amplified product fragments were recovered according to the instructions of the PCR Recovery Kit. The recovered amplified product fragment and the pMD18-T vector were incubated overnight at 16°C under the action of Ligation Solution I.

4. Transformation of E. coli

Escherichia coli was transformed by calcium chloride method, and positive clones of transformed E. coli were detected by blue-white screening.

5. Detection

Using specific primers (svaL and svaR) to perform PCR detection on the screened E. coli positive clones, and extract the DNA of plasmid pMD-iac from the E. coli positive clones determined by PCR detection.

4. Sensitivity determination of template DNA

1. Quantitative determination of amplified internal standard and pure cultured total DNA of Salmonella

The contents of the purified amplified internal standard and the purely cultured total DNA of Salmonella were 11.9 ng/μL and 12.8 ng/μL, respectively, as measured by a DU-800 ultraviolet spectrophotometer (Beckman Coulter). According to the calculation formula Ncopies/μL=mass of PCR fragment (g/μL)/(660g/mol×number of bases)×(6.023×10 ²³ ), the copy number of 1 μL amplified internal standard can be obtained as 3.65×10 ⁹ .

2. Detection sensitivity when no amplification internal standard is added

The above-mentioned total DNA solution of Salmonella choleraesuis determined above was diluted 10 times with sterile water, and 5 μL was respectively taken from the 11.9ng/μL-1.19fg/μL DNA solution and added to the PCR reaction system, so that each PCR reaction system (25 μL) Containing DNA respectively: 2.38ng/μL, 238pg/μL, 23.8pgμL, 2.38pg/μL, 238fg/μL, 23.8fg/μL, 2.38fg/μL, 0.238fg/μL. The amplification results are shown in Figure 1, only the PCR reaction system containing 0.238fg/μL DNA did not have the target sequence amplification band, while the other PCR reaction systems all had the target sequence amplification band. Therefore, its detection sensitivity is 2.38fg/μL (starting concentration is 11.9fg/μL)

3. Detection sensitivity after adding internal standard for amplification

The purified amplified internal standard was diluted 10-fold with sterile water, from 3.65×10 ⁹ copies/μL to 3.65 copies/μL. Then, the amplified internal standard was added to the above-mentioned PCR detection system to study the influence of the amplified internal standard on the sensitivity. When the PCR reaction system adds 3.65×10 ⁴ copies of internal standard, its detection sensitivity is still 12.8 fg/μL (as shown in FIG. 2 ). When the amount of amplified internal standard added to the PCR reaction system is less than 3.65×10 ⁴ copies, because the fluorescence intensity of the amplification product of the amplified internal standard is weak, it cannot reach the purpose of indicating a false negative result and is not used. Therefore, adding 3.65×10 ⁴ copies of the amplified internal standard has almost no effect on the sensitivity of the above PCR detection system. In the PCR detection system established in this study, 3.65×10 ⁴ copies of the amplified internal standard were used.

4. Detection of colony sensitivity

Pick Salmonella choleraesuis from the test tube slant or plate and insert it into BPW (buffered peptonewater) liquid medium. After 8 hours of enrichment, make 10-fold gradient dilution with normal saline, and dilute 10 gradients in total. Counting method to calculate the colony concentration of Salmonella. After calculation, the colony concentration of Salmonella in BPW liquid medium was 3.7×10 ⁸ cfu/mL. After PCR amplification, the detectable colony sensitivity was 3.7×10 ³ cfu/mL (as shown in FIG. 3 ).

5. Specific detection

The PCR detection system established in this study was used to amplify and detect 44 strains of common Salmonella and 22 strains of non-Salmonella, including Salmonella typhi, Salmonella paratyphi A, and Salmonella choleraesuis, and found that all of them could be amplified using the genomic DNA of Salmonella as a template A target sequence-specific product of 364bp was produced, while only an internal standard of 516bp could be amplified when non-Salmonella genomic DNA was used as a template.

6. Anti-interference detection

Proteus mirabilis (P.mirabilis), Escherichia coli (E.coli), and Staphylococcus aureus (S.aureus) were used as interfering strains to examine the effect on colony sensitivity of Salmonella choleraesuis (S.choleraesuis AS1.1190) . Single colonies of Proteus mirabilis, Escherichia coli, Staphylococcus aureus and Salmonella choleraeesuis were picked from the plate, respectively inserted into 50mL BPW liquid medium, and cultured at 37°C for 6h. Then use sterilized physiological saline to make 10-fold serial dilutions and dilute to 10 ^-10 , and take 1mL of the bacterial solution with a dilution of 10 ^-6 and 10 ^-7 for plate counting, and count Proteus mirabilis, Escherichia coli, golden yellow Initial concentrations of pure cultures of Staphylococcus and S. choleraesuis, respectively. The initial concentrations of Proteus mirabilis, Escherichia coli, Staphylococcus aureus and Salmonella choleraesuis were calculated to be 2.1×10 ⁸ , 1.2×10 ⁸ , 3.1×10 ⁸ and 3.7×10 ⁸ .

At the same time, take 1 mL of Salmonella choleraesuis 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 times diluted The bacterial solution was added to a test tube containing 6 mL of BPW liquid medium, and two sets of repetitions were made. Then, take 1 mL of N×10 ⁴ , N×10 ⁶ and N×10 ⁸ cfu/mL bacterial suspensions of Proteus mirabilis, Escherichia coli and Staphylococcus aureus and add them to three groups of test tubes respectively (as shown in Figure 4 ). After the bacterial solution in the test tube was evenly mixed with the medium, 1 mL was sampled from each test tube, stored in a 1.5 mL centrifuge tube, DNA was extracted and placed at 4°C for later use. Then put the 3 groups of test tubes in a shaker at 37°C and incubate them at 150rpm for 8h, sample 1mL from each test tube again, store in a 1.5mL centrifuge tube, extract and store at 4°C for use.

The results of PCR detection test showed that the interfering strains did affect the sensitivity detection of Salmonella colonies, but after 12 hours of enrichment, the interference was reduced, and the initial amount of Salmonella could be detected at 3-4 cfu/mL.

7. Accuracy evaluation

1. Detection of artificially contaminated samples

Collect 80 milk samples with severe artificial contamination, take 25mL of each sample, transfer to 225mL BPW liquid medium, and enrich the bacteria at 37°C for 8h. Salmonella DNA was extracted for PCR detection, and 77 of the test results were positive, and 3 were false negative (amplified products without target sequence and internal standard). And when the same template DNA is detected by a PCR system without an internal standard for amplification, the detection result is consistent with the above. The false-negative samples were tested again after the DNA was re-purified, and the false-negative samples were all shown as positive results. This shows that there are interfering factors in the template DNA solution extracted from these three false negative samples. The detection system established in this study can indeed indicate false negatives when testing a large number of samples, which helps to improve the accuracy of detection.

2. Detection of natural contamination samples

50 eggs were collected, and three methods were used to test the samples at the same time, namely PCR detection, national standard detection and API reagent strip detection.

Firstly, take 25mL of each egg, transfer it into 25mL sterilized normal saline, homogenate, take 25mL sample homogeneous solution and add 100mL Magnesium Chloride Malachite Green Enrichment Solution (MM) and 100mL Nitrite Cystine Enrichment Solution (SC) Carry out selective enrichment culture. After the samples inserted into the nitrite cystine enrichment solution were incubated at 36°C for 8 hours, 1 mL was sampled from each sample for PCR detection. The PCR test results showed that only 5 positive control samples were positive, and the rest of the samples were all negative.

Then, after 24 hours of selective enrichment culture, they were separated on bismuth sulfite (BS) agar plate and DHL agar plate, and suspicious colonies were taken from the two selective plates, and further detected and identified by biochemical tests and API reagent strips. . The biochemical identification results of the national standard test and the test results of the API reagent strip showed the same results as the PCR test, that is, only 5 positive samples were detected with Salmonella, and most of the remaining suspicious colonies selected were E. coli and citric acid bacteria.

The results of the three tests are completely consistent, indicating that the detection accuracy of the PCR detection system established in this study is consistent with the national standard detection method and the API reagent strip detection method.

Claims (10)

1, a kind of Salmonella PCR detection method that has added amplification internal standard, it is characterized in that, concrete steps are as follows: (1) Using the specific gene sequence of Salmonella itself to construct an internal standard for amplification and design specific primers; (2) First, according to the Tm value recommended when designing the primers, set the temperature gradient at intervals of 1°C within the range of Tm±5°C, and select the annealing temperature according to the brightness of the amplified product in the electrophoretic graph. After determining the annealing temperature, Select the Mg ^{concentration} of the PCR reaction system in the reaction system; (3) Carry out PCR detection, select the detection sensitivity of PCR, then, take the carrier with the amplification internal standard, add in the PCR reaction system, add the amplification internal standard of different dilution concentrations in the reaction system, again, detect DNA or bacterial strain The sensitivity of the amplification internal standard that has the least impact on the detection sensitivity and can clearly indicate false negatives is selected as the added concentration; (4) extract the DNA of the Salmonella standard strains of different serotypes and the non-Salmonella standard strains of different species, carry out PCR detection, determine whether the specificity of the primers and the amplification internal standard can be amplified normally in the non-Salmonella sample detection; (5) Judging whether the amplification internal standard indicates a false negative by detecting artificially polluted samples; testing natural polluted samples, and using the national standard method and the test results of API reagent strips for reference to evaluate whether the test results of the established PCR detection system are accurate . 2. The method for detecting Salmonella PCR with an amplified internal standard according to claim 1, wherein said selection of the Mg ^{concentration} of the PCR reaction system refers to: setting an interval of 0.5mmol/L in the reaction system The Mg ²⁺ concentration gradient is from 1.0mmol/L to 3.0mmol/L, and the Mg ²⁺ concentration is also selected according to the brightness of the amplified product in the electropherogram. 3. The method for detecting Salmonella PCR with an amplified internal standard according to claim 1, wherein the detection sensitivity of the selective PCR refers to that the measured Salmonella total DNA solution is made 10 times higher with sterile water. Gradual dilution to 10 ^-10 , using the diluted DNA solution as a template, 5 μL was added to the PCR reaction system for PCR detection, and sterile water was used instead of the DNA template as a negative control, and then the detection sensitivity of PCR was selected. 4. The Salmonella PCR detection method with an amplified internal standard according to claim 1, characterized in that the detection of artificially contaminated samples refers to: collecting 80 milk samples with serious artificial contamination, each taking 25mL , transferred to 225mL of BPW liquid medium, enriched in a shaker at 37°C, and took a sample after 8 hours of enrichment, 1mL of each sample was taken, put into a 1.5mL centrifuge tube, centrifuged at 3000r/min for 10min, and precipitated. food residues in the food. Take the supernatant, centrifuge at 12000r/min for 5min, collect the Salmonella cells, pour off the supernatant, resuspend the cells with sterile double distilled water, and wash by centrifugation three times. Then add 100 μL of sterile ultrapure water, cook in a boiling water bath for 10 minutes, take it out of the boiling water bath, immediately place it at -20°C for 30 minutes, and after thawing at 37°C, centrifuge at 12,000 r/min for 5 minutes, and the supernatant is PCR template DNA Solution, 5 μL was added to the PCR reaction system for PCR detection, and sterile water was used instead of the DNA template as a negative control. 5. The method for detecting Salmonella PCR with an amplified internal standard according to claim 1, characterized in that the detection of the naturally contaminated food samples refers to: 50 eggs were collected, and 25mL of each egg was transferred into In 25mL sterilized normal saline, homogenate, take 25mL sample homogeneous liquid into 100mL magnesium chloride malachite green enrichment solution and 100mL nitrite cystine enrichment solution for selective enrichment culture, add nitrite cystine After the samples of the enrichment solution were cultured at 36°C for 8 hours, 1 mL was sampled from each sample for PCR detection. After 24 hours of selective enrichment culture, they were then separated on bismuth sulfite agar plates and DHL agar plates. Suspicious colonies were collected on the positive plate, and further detected and identified by biochemical tests and API reagent strips. 6. The method for detecting Salmonella PCR with an internal standard for amplification according to claim 1, characterized in that said construction of an internal standard for amplification refers to: utilizing the specific gene sequence of Salmonella itself to construct an internal standard for amplification, reducing It avoids the interference of DNA from organisms other than Salmonella on PCR detection, and also avoids the interference of homologous cross-linking of the amplified internal standard on the target gene. The amplified internal standard reduces the interference on the target gene during the detection process and increases the accuracy of detection. 7. According to claim 1 or 6, the method for detecting Salmonella PCR with an internal standard for amplification is characterized in that the internal standard for amplification and the method for amplification are as follows: using sequence analysis and gene comparison The method selects the specific gene sequence used to construct the amplified internal standard for Salmonella PCR detection, and has a very low homology with the target gene to be detected; the two ends of the selected gene sequence are respectively connected with detection primers by means of gene cloning to construct The amplified internal standard was cloned into the vector; the above-mentioned vector with the amplified internal standard was transferred to E. coli by transgenic method, and then spread on a selective plate and incubated at 37°C for 12 hours. Use a sterilized toothpick to pick the white colony from the selective plate, inoculate it into a test tube containing 5ml of LB culture solution, and then culture it at 150r/min at 37°C for 6h, extract the DNA of plasmid pMD-stn, and perform PCR After amplification, it was confirmed that transformants were obtained. 8. The method for detecting Salmonella PCR with an internal standard for amplification according to claim 1, wherein said specific primers refer to: a pair of detection primers used for PCR detection of Salmonella, when performing PCR detection, It can amplify the specific detection gene of Salmonella strains, or the amplification internal standard added in the PCR system; there are also two pairs of amplification primers and long primers used to construct the amplification internal standard, and the amplification primers can only amplify The DNA fragments used to construct the amplified internal standard are obtained, and the long primers are used to obtain the amplified internal standard by means of PCR amplification and gene cloning. 9. The method for detecting Salmonella by PCR with amplified internal standard according to claim 1 or 7, characterized in that the vector, the host cell transformed by it, contains the DNA fragment of the amplified internal standard. 10. The method for detecting Salmonella PCR with an amplified internal standard according to claim 1, wherein said false negative means that the PCR reaction is affected by the inhibitors present in food or medium and other substances. No reaction occurred, or the result was negative due to operator error.

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