NL2036756A

NL2036756A - Fluorescent pcr method for detecting listeria monocytogenes and mycobacterium tuberculosis in milk

Info

Publication number: NL2036756A
Application number: NL2036756A
Authority: NL
Inventors: Zhang Wei; Zhang Hui; Liu Feifan; Liu Liangbo; Shi Chao; Deng Xingmei; Wang Zhen; Guo Jia; Zhou Xia; Sun Zhihua
Original assignee: Univ Shihezi
Priority date: 2024-01-05
Filing date: 2024-01-05
Publication date: 2024-01-30
Also published as: NL2036756B1

Abstract

The present invention provides a fluorescent PCR method for detecting Listeria monocytogenes and Mycobacterium tuberculosis in milk, and relates to the field of PCR detection. The method includes the following steps: Sl. determination of fluorescent 5 quantitative PCR specific targets; 82. primer and, probe design labeling and synthesis; S3. strain. activation; S4. genomic DNA extraction; S5. standard, plasmid construction; 86. primer concentration optimization; 87. probe concentration optimization; and 88. standard curve plotting. The establishment of etiological lO detection method for Listeria monocytogenes and Mycobacterium tuberculosis provides an auxiliary means for detection of Listeria monocytogenes and Mycobacterium tuberculosis in raw milk. (+ Fig. l) 15

Description

P1961 /NLpd

FLUORESCENT PCR METHOD FOR DETECTING LISTERIA MONOCYTOGENES AND

MYCOBACTERIUM TUBERCULOSIS IN MILK

Technical field

The present invention relates to the technical field of PCR detection, in particular to a fluorescent PCR method for detecting

Listeria monocytogenes and Mycobacterium tuberculosis in milk.

Background technology

Milk is the most consumed natural beverage worldwide due to high quality protein and nutritional ingredients. Zoonosis, an in- fectious disease caused by zoonotic pathogens, can spread between humans and animals through direct or indirect contact, and the consumption of insufficiently cooked or unautoclaved food of ani- mal origin, such as meat, milk, and products thereof, is an im- portant way to spread zoonotic diseases. Raw milk is a major fac- tor in spreading foodborne pathogens through milk and dairy prod- ucts.

A wide range of microorganisms and a variety of pathogenic bacteria are present in raw milk. Listeria monocytogenes, as an important foodborne pathogen, can survive and multiply in low- temperature environments where the milk and products thereof are often saved, significantly increasing the risk of spread. Current- ly, the detection of Listeria monocytogenes and Mycobacterium tu- berculosis in milk and products thereof can be achieved by tradi- tional isolated culture, immunological detection, and molecular biological detection. The traditional isolated culture requires bacterial isolation and purification, which is time-consuming and low sensitive, and is not applicable to slow-growing bacteria such as Mycobacterium tuberculosis. Immunological methods are based on the specific binding between antigens and antibodies, but due to the inherent problems of milk, there may not be a desirable detec- tion effect when utilizing immunological methods, which limits the use of immunological methods to a certain extent.

Summary

Aiming at the deficiencies of the prior art, the present in-

vention provides a fluorescent PCR method for detecting Listeria monocytogenes and Mycobacterium tuberculosis in milk, and solves the problem that traditional isolated culture requires separation and purification of bacteria, which is time-consuming and low sen- sitivity.

In order to realize the above objective, the present inven- tion provides the following technical solutions: a fluorescent PCR method for detecting Listeria monocytogenes and Mycobacterium tu- berculosis in milk includes the following steps:

S1. determination of fluorescent quantitative PCR specific targets taking whole gene sequences of a Listeria monocytogenes standard strain IM EDG-e and a Mycobacterium tuberculosis standard strain H37Rv as references, determining real-time fluorescence quantitative PCR targets for detecting Listeria monocytogenes and

Mycobacterium tuberculosis as an lmo0733 gene and an Rv2875 gene separately by comparing with BLAST of an NCBI database, and per- forming homology analysis on the 1mo0733 and the Rv2875 genes;

S2. primer and probe design labeling and synthesis taking the lmo0733 gene of Listeria monocytogenes and the

Rv2875 gene of Mycobacterium tuberculosis as targets, and perform- ing primer and probe design for real-time fluorescence quantita- tive PCR by reference to the design principle of TagMan probe method;

S3. strain activation a. activation of Listeria monocytogenes and other strains weighing 3.85 g of BHI powder, dissolving the same with water and fixing a solution to 100 mL, preparing a BHI liquid culture medium after autoclaving and drying the solution, taking 20 pL of preserved Listeria monocytogenes LM90, LMEDG-e, Salmonella, Staph- ylococcus chromogenes, Staphylococcus lentus, Staphylococcus homi- nis, Staphylococcus aureus and Escherichia coli to be cultured in a 20 mL BHI liquid culture medium on a shaking table at 170 rpm and at 37°C for 16-18 h, weighing 3.0 g of TSB powder, dissolving the same with water and fixing a solution to 100 mL, preparing a

TSB liquid culture medium after sub-packaging, autoclaving and drying the solution, separately taking 20 pL of preserved Strepto-

coccus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis to be inoculated in a 20 mL TSB liquid culture medium for culturing on a shaking table at 170 rpm and at a constant tempera- ture of 37°C horizontally for 24-48 h; and b. preparation of modified neutral Lowenstein-Jensen culture medium and activation of Mycobacterium tuberculosis autoclaving a Lowenstein-Jensen culture medium by reference to the method described by Ji Xiang, placing a prepared culture medium into a 37°C constant temperature incubator for culturing for 48 h to test whether contamination exists, preserving a pollution- free culture medium at 4°C away from light for later use, taking, with an aseptic inoculation loop, a loop of Mycobacterium bovis

BCG preserved in the experiment from an ultra-clean workbench to inoculate on a surface of the modified neutral Lowenstein-Jensen culture medium, and placing the medium in a 37°C constant tempera- ture incubator for culturing for 4-6 weeks;

S4. genomic DNA extraction inactivating an activated strain in a water bath at 80°C for 30 min, collecting bacteria by centrifugation at 12000 rpm, and performing the subsequent extraction of bacterial genomic DNAs ac- cording to the instructions of a DNA extraction kit;

S5. standard plasmid construction taking extracted genomic DNAs of the Listeria monocytogenes

LM90 or the Mycobacterium bovis BCG as templates, amplifying gPCR target gene fragments using upstream and downstream primers of qPCR, verifying PCR products by 2% agarose gel electrophoresis (AGE) and then recovering a target band, ligating a ligation sys- tem to a pMD19-T cloning vector at 16°C overnight, transforming a ligation product according to the operation instructions of DHbwo competent cells, coating the transformed ligation product on an

Amp-resistance plate, culturing at a constant temperature of 37°C overnight, selecting single colonies and performing bacteria solu- tion PCR verification next day, and preserving bacteria of the correctly verified recombinant plasmids and sending the plasmids to the company for sequencing;

S6. primer concentration optimization taking the extracted genomic DNAs from the Listeria monocyto- genes LM90 or from the Mycobacterium bovis BCG as templates, opti- mizing a primer concentration in a reaction system by setting a final primer concentration gradient of 0.2 pM/L, 0.4 uM/L, 0.6 uM/L, 0.8 uM/L, and 1.0 pM/L, and screening and determining an op- timal primer concentration based on the principle of a minimum CT value and a maximum ARn value;

S7. probe concentration optimization optimizing, under the initial reaction system and the reac- tion procedure as described above, a probe concentration in the reaction system by setting a final concentration of the probe at 50 nM/L, 100 nM/L, 150 nM/L, 200 nM/L ‚and 250 nM/L, and screening and determining an optimal probe concentration based on the prin- ciple of a minimum CT value and a maximum ARn value;

S8. standard curve plotting activating, in 20 mL of LB broth containing Amp-resistance, the recombinant plasmids that verified as correct after sequenc- ing, collecting the precipitation of a bacterial solution by cen- trifugation, extracting and purifying the recombinant plasmids, and calculating a copy number after determining the concentration of the recombinant plasmids, diluting the recombinant plasmids in a 10-fold gradient and performing qPCR reaction using each dilu- tion as a template, plotting a standard curve according to the re- lationship between a CT value and a logarithmic value of the copy number of the plasmid, and calculating an amplification efficiency of the gPCR reaction using a qPCR reaction efficiency calculation formula;

S9. evaluation of the specificity of fluorescent quantitative

PCR for Listeria monocytogenes and Mycobacterium tuberculosis carrying out PCR reaction using extracted genomic DNAs of the Listeria monocytogenes LM90, the LM EDG-e, the Mycobacterium bovis BCG, the Salmonella, the Staphylococcus chromogenes, the

Staphylococcus lentus, the Staphylococcus aureus, the Staphylococ- cus hominis, the Streptococcus uberis, the Streptococcus agalac- tiae, the Streptococcus dysgalactiae, the Escherichia coli and the

Enterococcus hirae as templates, and setting up ddH:0 as a negative control for the templates, and determining the result by observing an amplification curve and the CT value; and

S10. evaluation of the sensitivity of fluorescent quantita- tive PCR for Listeria monocytogenes and Mycobacterium tuberculosis carrying out a 10-fold gradient dilution on recombinant 5 standard plasmids pMD19-T-1mo0733 and pMD13-T-Rv2875 with known copy numbers, and carrying out PCR reaction with each dilution of the plasmid as a template, with ddH,0 as a negative control, and performing conventional PCR to analyze the sensitivity of qPCR.

Preferably, in S3, the raw materials of the Lowenstein-Jensen culture medium include fresh whole egg liquid, 2% malachite green liquid, glycerol, magnesium sulfate, sodium glutamate, potassium dihydrogen phosphate, magnesium citrate and deionized water.

The present invention provides a fluorescent PCR method for detecting Listeria monocytogenes and Mycobacterium tuberculosis in milk and has the following advantageous effects. 1. According to the present invention, an etiological detection method for Listeria monocytogenes in raw milk is established. A fluorescent PCR detection method based on the specific genes of

Listeria monocytogenes and Mycobacterium tuberculosis is estab- lished. The establishment of etiological detection method for Lis- teria monocytogenes and Mycobacterium tuberculosis provides an auxiliary means for detection of Listeria monocytogenes and Myco- bacterium tuberculosis in raw milk, and a dual gPCR method for de- tecting Listeria monocytogenes and Mycobacterium tuberculosis in milk is established, realizing the detection of two target patho- gens in the same reaction system. At the same time, sample DNA ex- traction, gPCR detection and result analysis can be achieved in 1.5 h.

Brief description of the drawings

FIG. 1 is a schematic diagram of a procedure of a reaction system according to the present invention;

FIG. 2 is a schematic diagram of a procedure of PCR reaction according to the present invention;

FIG. 3 is a schematic diagram of the optimization of a primer concentrationof an lmo0733 gene according to the present inven- tion;

FIG. 4 is a schematic diagram of the optimization of a probe concentration of the 1lmo0733 gene according to the present inven- tion;

FIG. 5 is a schematic diagram of PCR target fragment ampli- fication of the 1mo0733 gene according to the present invention;

FIG. 6 is a schematic diagram of PCR verification of a bacte- rial solution according to the present invention;

FIG. 7 is a diagram showing a comparison result for a se- quenced sequence of pMD1%-T-1mo0733 recombinant plasmids according to the present invention; and

FIG. 8 is a schematic diagram of a qPCR standard curve of

Listeria monocytogenes according to the present invention.

Detailed description

The technical solutions of the examples in the present inven- tion will be described clearly and completely by reference to the accompanying drawings of the examples in the present invention be- low. Obviously, the examples described are only some, rather than all examples of the present invention. Based on the examples of the present invention, all the other examples obtained by those ordinary skilled in the art without creative efforts fall within the scope of protection of the present invention.

Examples

As shown in FIGS. 1-8, an example of the present invention provides a fluorescent PCR method for detecting Listeria monocyto- genes and Mycobacterium tuberculosis in milk, including the fol- lowing steps.

S1. Determination of fluorescent quantitative PCR specific targets whole gene sequences of a Listeria monocytogenes standard strain LM EDG-e and a Mycobacterium tuberculosis standard strain

H37Rv were taken as references, real-time fluorescence quantita- tive PCR targets for detecting Listeria monocytogenes and Mycobac- terium tuberculosis were determined as an lmo0733 gene and an

Rv2875 gene separately by comparing with BLAST of an NCBI data- base, and homology analysis was performed on the 1lmo0733 and the

Rv2875 genes.

S2. Primer and probe design labeling and synthesis the 1mo0733 gene of Listeria monocytogenes and the Rv2875 gene of Mycobacterium tuberculosis were taken as targets, and pri- mer and probe design for real-time fluorescence quantitative PCR were performed by reference to the design principle of TagMan probe method. The primer and probe sequences are shown in Table 1 below.

Table 1 gPCR primer and probe for Listeria monocytogenes and

Mycobacterium tuberculosis

Name Sequence {5'-3') {bp) modification

Îmo0733-F GACTGTCAGTTCCTGATTCCGATAT ~~ Non

Imo0733-R CCATAACAGTTTCTCCAAGCAAAG 90 Non

Imo0733-P ATCTCCATATCGGAAGTGCTTGAAACACCA 5'FAM, 3'BHQ1

Rv2875-F CAGCTCAATCCGCAAGTAA Non

Rv2875-R CCGGCAGCTTGCTAAAT 94 Non

Rv2875-P CACCCTCAACAGCGGTCAGTACAC 5'VIC, 3'BHQ1 a. activation of Listeria monocytogenes and other strains 3.85 g of BHI powder was weighted and dissolved with water, a solution was fixed to 100 mL, a BHI liquid culture medium was pre- pared after autoclaving and drying the solution, and 20 uL of pre- served Listeria monocytogenes LM90, LMEDG-e, Salmonella, Staphylo- coccus chromogenes, Staphylococcus lentus, Staphylococcus hominis,

Staphylococcus aureus and Escherichia coli was taken to be cul- tured in a 20 mL BHI liquid culture medium on a shaking table at 170 rpm and at 37°C for 16-18 h; and 3.0 g of TSB powder was weighted and dissolved with water, a solution was fixed to 100 mL, a TSB liquid culture medium was prepared after sub-packaging, au- toclaving and drying the solution, and 20 uL of preserved Strepto- coccus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis were separately taken to be inoculated in a 20 mL TSB liq- uid culture medium for culturing on a shaking table at 170 rpm and at a constant temperature of 37°C horizontally for 24-48 h. b. Preparation of modified neutral Lowenstein-Jensen culture medium and activation of Mycobacterium tuberculosis a Lowenstein-Jensen culture medium was autoclaved according to the method described by Ji Xiang. A prepared culture medium was placed into a 37°C constant temperature incubator for culturing for 48 h to test whether contamination exists; a pollution-free cul- ture medium was preserved at 4°C away from light for later use; and a loop of Mycobacterium bovis BCG preserved in the experiment was taken from an ultra-clean workbench with an aseptic inoculation loop to inoculate on a surface of the modified neutral Lowenstein-

Jensen culture medium, and the medium was placed in a 37°C constant temperature incubator for culturing for 4-6 weeks. 34. Genomic DNA extraction an activated strain was inactivated in a water bath at 80°C for 30 min, and was centrifugated at 12000 rpm to collect bacte- ria, and the subsequent extraction of bacterial genomic DNAs was carried out according to the instructions of a DNA extraction kit. 35. Standard plasmid construction the extracted genomic DNAs from the Listeria monocytogenes

LM90 or from the Mycobacterium bovis BCG were taken as templates, and gPCR target gene fragments were amplified using upstream and downstream primers of PCR. The reaction system is shown in Table 2, and the procedure is shown in FIG. 1. PCR products were veri- fied by 2% agarose gel electrophoresis (AGE) and then a target band was recovered, and ligation systems shown in Table 3 were li- gated to a pMD19-T cloning vector at 16°C overnight; ligation prod- ucts were transformed according to the operation instructions of

DHSa competent cells and the transformed ligation products were coated on an Amp-resistance plate for culturing at a constant tem- perature of 37°C overnight; single colonies were selected and bac- teria solution PCR verification was performed next day, and bacte- ria of the correctly verified recombinant plasmids were preserved and the plasmids were sent to a company for sequencing. The re- sults of PCR amplification of the target gene showed that the tar- get band produced by amplification was single with an expected size, as shown in FIG. 5, where M was the 100bp Ladder; 1-3 was the amplification of the gPCR target fragment of the 1mo0733 gene; and 4 was the negative control. The PCR results of the bacterial solution showed that the monoclonal colonies were all positive clones, as shown in FIG. 6, where M was the 100bp Ladder; 1-6 were positive clones; and 7 was the negative control. The positive clones verified correctly were sequenced by the company, and the sequencing results were compared with the lmo0733 gene of LM EDG- e, and some of the compared sequences are shown in FIG. 7, and the identity between the sequenced sequence and the reference sequence was 100%.

Table 2 PCR reaction system

Component Volume 2xTaq Master PCRMix Sl

F (10 uM) Tul

R (10 uM) 1ub

Template 2ul ddH,0 8.5ulL

Total 25 ut

Table 3 Ligation reaction system

Component Volume “solutiont 45m pMD 19-T Vector 0.5uL

Gel recovery products 5ut

Total 10 HL 86. Primer concentration optimization the extracted genomic DNAs from the Listeria monocytogenes

LM90 or from the Mycobacterium bovis BCG were taken as templates according to the instructions of the HyperProbe Mixture from Com-

Win Biotech Co.,Ltd. The initial reaction system is shown in Table 4 and the reaction procedure is shown in FIG. 2. A primer concen- tration in the reaction system was optimized by setting a final primer concentration gradient of 0.2 uM/L, 0.4 wM/L, 0.6 uM/L, 0.8 uM/L, and 1.0 uM/L (the corresponding relationship between the fi- nal concentration of primer and the amount added in the reaction system is shown in Table 5), and an optimal primer concentration was screened and determined based on the principle of a minimum CT value and a maximum ARn value. The results showed that the CT val- ue was the smallest and the ARn value was the largest when the fi- nal primer concentration was at 0.4 um/L, as shown in FIG. 3,

where the primer concentrations of 1-5 were 0.2 uM/L, 0.4 uM/L, 0.6 yM/L, 0.8 uM/L, and 1.0 pM/L, respectively, and thus, 0.4 uM/L was selected as the optimal primer concentration.

Table 4 gPCR reaction system

Component Amount added in 20 pi system “2xHyperProbe Mixture 125.0

F {10 uM/L) 0.4 ul

R {10 uM/L) 0.4 ul

Probe 0.4 ul

Template X ub ddH,0 Replenishment to 20 ub

Table 5 The corresponding relationship between the final con- centration of primers and the amount added in the reaction system

Final concentration of primers (4M/L) Addition 0.2 0.4 HL 0.4 0.8 ul 0.6 1.2 ul 0.8 1.6 HL 1.0 2.0 4L 87. Probe concentration optimization under the initial reaction system and the reaction procedure as described above, a probe concentration in the reaction system was optimized by setting a final concentration of the probe at 50 nM/L, 100 nM/L, 150 nM/L, 200 nM/L ‚and 250 nM/L (the correspond- ing relationship between the final concentration of the probe and the amount added in the reaction system is shown in Table 6), and an optimal probe concentration was screened and determined based on the principle of a minimum CT value and a maximum ARn value.

The results showed that the CT value was the smallest and the ARn value was the largest when the final concentration of the probe was at 250 nM/L, as shown in FIG. 4, where the concentrations of primers 1-5 were 0.2 pM/L, 0.4 uM/L, 0.6 uM/L, 0.8 uM/L, and 1.0 uM/L, respectively, and thus, 250 nM/L was selected as the optimal probe concentration.

Table 6 The corresponding relationship between the final con-

centration of probes and the amount added in the reaction system

Final concentration of probes (nM/L) Addition 50 0.1puL 100 0.2 ul 150 0.3 HL 200 0.4 ul 250 0.5 HL

SB, standard curve plotting the recombinant plasmids that verified as correct after se- quencing were activated in 20 mL of LB broth containing Amp- resistance; the precipitation of a bacterial solution was collect- ed by centrifugation, the recombinant plasmids were extracted and purified, and a copy number was calculated after determining the concentration of the recombinant plasmids; the recombinant plas- mids were diluted in a 10-fold gradient and gPCR reaction was per- formed using each dilution as a template; a standard curve was plotted according to the relationship between the CT value and a logarithmic value of the copy number of the plasmid, and an ampli- fication efficiency of the gPCR reaction was calculated using a qPCR reaction efficiency calculation formula; the pMD 19-T-1mo0733 recombinant plasmid was successfully constructed, and plasmid ex- traction was carried out after activation in an Amp-resistance LB liquid medium, and the concentration of the recombinant plasmid was measured to be 422.6 ng/pL, with the size of 2782 bp, and a copy number of 1.39x10"* copies/uL. The recombinant plasmid was di- luted with ddH,0 to prepare a gradient of 1.39x10° copies/uL or 1.39x10" copies/uL and gPCR reaction was performed. Taking the log- arithmic value of the copy number of the recombinant plasmid as a horizontal coordinate and the CT value as a vertical coordinate, the standard curve was plotted using Graphpad prism, and the re- sults are shown in FIG. 8. The equation of the standard curve was y = -3.3635x + 43.761 with R® = 0.9909, which showed a good linear relationship. The amplification efficiency calculated according to the slope of the standard curve was 94.03%, which was in a reason- able range, proving that the design of probe and primer was rea- sonable and the reaction conditions were well optimized.

S59. Evaluation of the specificity of fluorescent quantitative

PCR for Listeria monocytogenes and Mycobacterium tuberculosis gPCR reaction was carried out using the extracted genomic

DNAs of the Listeria monocytogenes LM90, the LM EDG-e, the Myco- bacterium bovis BCG, the Salmonella, the Staphylococcus chromo- genes, the Staphylococcus lentus, the Staphylococcus aureus, the

Staphylococcus hominis, the Streptococcus uberis, the Streptococ- cus agalactiae, the Streptococcus dysgalactiae, the Escherichia coli and the Enterococcus hirae as templates, ddH-0 was set up as a negative control for the templates, and the result could be deter- mined by observing an amplification curve and the CT value. Only the presence of the target strains (Listeria monocytogenes LM90 and LM EDG-e) produced significant amplification and the typical

S-shaped curve. As for non-target strains such as the Salmonella,

Staphylococcus ( the Staphylococcus chromogenes, the Staphylococ- cus lentus, the Staphylococcus aureus, and the Staphylococcus hom- inis), Streptococcus (the Streptococcus uberis, the Streptococcus agalactiae, and the Streptococcus dysgalactiae), the Escherichia coli, and the Enterococcus hirae, there was no significant ampli- fication of gPCR, indicating that gPCR primers and probes designed based on the Listeria monocytogenes 1lmo0733 gene have good speci- ficity. 310. Evaluation of the sensitivity of fluorescent quantita- tive PCR for Listeria monocytogenes and Mycobacterium tuberculosis a 10-fold gradient dilution was carried out on recombinant standard plasmids pMD19-T-1mo0733 and pMD19-T-Rv2875 with known copy numbers, and gPCR reaction was carried out with each dilution of the plasmid as a template, with ddH:0 as a negative control, and conventional PCR was carried out to analyze the sensitivity of

QPCR.

In 383, the raw materials of the Lowenstein-Jensen culture me- dium include fresh whole egg liquid, 2% malachite green liquid, glycerol, magnesium sulfate, sodium glutamate, potassium dihydro- gen phosphate, magnesium citrate and deionized water. The formula for the modified neutral Lowenstein-Jensen culture medium is shown in Table 7.

Formula Dosage mula) “Fresh whole egg liquid Freshegg ~~ 500mL 2% malachite green

Malachite green (C23H25C1N:) 20 mL liquid

Glycerol (C3HgO3) 6 mL

Magnesium sulphate (MgS0,) 0.128

Sodium glutamate (CsHsNO,Na) 3.66

Potassium dihydrogen phos-

Basic solution 1.28 phate(Na,HPO,);

Magnesium citrate 0.128 (C12H10Mg83014)

Deionized water 300 mi

A method for dual qPCR detection of Listeria monocytogenes and Mycobacterium tuberculosis includes the following steps. 81. Preliminary establishment

Double gPCR was performed on part of the primer concentra- tions and probes optimized in S5 and S6 above, the reaction system is shown in Table 8 below, and the reaction procedure was carried out as shown in FIG. 2.

Table 8 Dual qPCR reaction system for Listeria monocytogenes and Mycobacterium tuberculosis “Component Amount added in 40 pL system 0 “2xHyperProbe Mixture ~~ 2sul

Imo0733-F 0.8 uL

Imo0733-R 0.8 uL

Imo0733-P 0.5 HL

Rv2875-F 2.0uL

Rv2875-R 2.0ulL

Rv2875-P 0.5 ul

Template X ub ddH,0 Replenishment to 40 pb 52. Evaluation of specificity of dual qPCR for Listeria mono- cytogenes and Mycobacterium tuberculosis

The genomic DNAs of Listeria monocytogenes LM90 and LM EDG-e were separately mixed with the genomic DNAs of Mycobacterium bovis

BCG according to 1:1 for gPCR reaction. Dual PCR specificity ex- periments were carried out with the DNAs of other non-target strains (Salmonella, Staphylococcus chromogenes, Staphylococcus lentus, Staphylococcus aureus, Staphylococcus hominis, Streptococ- cus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae,

Escherichia coli, and Enterococcus hirae) and ddH:0 as controls, and the result was determined by the amplification curve and the

CT value.

S3. Evaluation of sensitivity of dual gPCR for Listeria mono- cytogenes and Mycobacterium tuberculosis

The pMD19-T-1mo0733 recombinant plasmid with the copy number of 1.39 x 10° copies/uL and the pMD19-T-Rv2875 recombinant plasmid with the copy number of 1.03 x 10° copies/uL were mized at 1:1 and then diluted in a 10-fold gradient, and the gPCR reaction was car- ried out with the 10-fold gradient-diluted plasmid as a template, and the sensitivity of the double gPCR was evaluated according to the results.

Although the examples of the present invention have been shown and described, for those ordinary skilled in the art, it can be understood as various changes, modifications, replacements and variations can be made on these examples within the principle and spirit of the present invention. The scope of the present inven- tion is defined by the attached claims and the equivalent thereof.

Claims

CONCLUSIONS

1. Fluorescent PCR method for detecting Listeria monocytogenes and Mycobacterium tuberculosis in milk, comprising the following steps:

Sl. determination of fluorescent quantitative PCR-specific targets starting from complete gene sequences of a Listeria monocytogenes standard strain LM EDG-e and a Mycobacterium tuberculosis standard strain H37Rv as references, determination of real-time fluorescent quantitative PCR targets for the detection of Listeria monocytogenes and Mycobacterium tuberculosis as a lmo0733 gene and a Rv2875 gene separately by comparison with BLAST of an NCBI database, and performance of homology analysis on the lmo0733 and Rv2875 genes;

S2. primer and probe design, labeling and synthesis, targeting the lmo0733 gene of Listeria monocytogenes and the Rv2875 gene of Mycobacterium tuberculosis and performing primer and probe design for real-time fluorescence quantitative PCR with reference to the design principle of the TagMan probe method;

S3. strain activation a. activation of Listeria monocytogenes and other strains weigh 3.85 g BHI powder, dissolve it with water and fix a solution to 100 mL, prepare a liquid BHI culture medium after autoclaving and drying the solution, take 20 uL preserved Listeria monocytogenes LM90, LMEDG-e, Salmonella, Staphylococcus chromagnes, Staphylococcus lentus, Staphylococcus hominis, Staphylococcus aureus and Escherichia coli to be cultured in a 20 mL BHI liquid culture medium on a shaking table at 170 rpm and 37°C for 16-18 hours, weigh 3.0 g of TSB powder, dissolve it with water and fix a solution to 100 mL, prepare a liquid TSB culture medium after subpacking, autoclave and dry the solution, take separately 20 µL of preserved Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis to inoculate into a 20 mL TSB liquid culture medium for cultivation on a shaking table at 170 rpm and a constant temperature of 37°C horizontally for 24- 48 hours; and B. preparation of a modified neutral Lowenstein-Jensen culture medium and activation of Mycobacterium tuberculosis autoclaving a Lowenstein-Jensen culture medium according to the method described by Ji Xiang, placing a prepared culture medium in an incubator at a constant temperature of 37°C for culture for 48 hours to test for contamination, store a contaminant-free culture medium at 4°C away from light for later use, with an aseptic inoculation loop a loop of the Mycobacteria stored in the experiment bovis BCG from an ultra-clean bench to inoculate onto a surface of the modified neutral Lowenstein-Jensen culture medium and place the medium in a constant temperature incubator at 37°C for 4-6 weeks to culture ;

S4. extraction of genomic DNA inactivating an activated strain in a water bath at 80°C for 30 minutes, collecting bacteria by centrifugation at 12000 rpm and then extracting bacterial genomic DNA according to the instructions of a DNA extraction kit;

S5. construction of standard plasmids, taking extracted genomic DNA of Listeria monocytogenes LM90 or Mycobacterium bovis BCG as templates, amplifying gPCR target gene fragments using upstream and downstream primers of qPCR, verifying PCR products by 2 % agarose gel electrophoresis (AGE) and then recovering a target band, ligating a ligation system to a pMD19-T cloning vector at 16°C overnight, transforming a ligation product according to the operating instructions of DH5a competent cells, coating the transformed ligation product on an Amp resistance plate for culture at a constant temperature of 37°C overnight, select individual colonies and perform PCR verification of the bacterial solution the next day, and retain bacteria from the correctly verified recombinant plasmids and the plasmids send to a company for sequencing

S6. Primer concentration optimization taking extracted genomic DNA of Listeria monocytogenes LM90 or Mycobacterium bovis BCG as templates, optimizing a primer concentration in a reaction system by setting a final primer concentration gradient of 0.2 M/L, 0 .4 uM/L, 0.6 pM/L, 0.8 pM/L and 1.0 uM/L, and the screening and determination of an optimal primer concentration based on the principle of a minimum CT value and a maximum ARn value;

S7. probe concentration optimization optimizing, under the initial reaction system and reaction procedure described above, a probe concentration in the reaction system by setting a final probe concentration to 50 nM/L, 100 nM/L, 150 nM/L , 200 nM/L and 250 nM/L, and screening and determining an optimal probe concentration based on the principle of a minimum CT value and a maximum ARn value;

S8. plot standard curve, activate, in 20 mL LB broth with Amp resistance, the recombinant plasmids verified as correct after sequencing, collect the precipitate of a bacterial solution by centrifugation, extract and purify the recombinant plasmids, and calculating a copy number after determining the concentration of the recombinant plasmids, diluting the recombinant plasmids in a 10-fold gradient and performing a qPCR reaction using each dilution as a template, plotting a standard curve according to the relationship between a CT value and a logarithmic value of the copy number of the plasmid and calculating an amplification efficiency of the gPCR reaction using a PCR reaction efficiency calculation formula;

S9. evaluating the specificity of fluorescent quantitative PCR for Listeria monocytogenes and Mycobacterium tuberculosis performing a gPCR reaction with extracted genomic DNA of Listeria monocytogenes LM90, LM EDG-e, Mycobacterium bovis BCG, Salmonella, Staphylococcus chromogenes, Staphylococcus Jentus, Staphylococcus aureus, Staphylococcus hominis, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Enterococcus hirae as template

nen, and setting ddH:0 as a negative control for the templates, and determining the result by observing an amplification curve and the CT value; and

S10. evaluate the sensitivity of fluorescent quantitative PCR for Listeria monocytogenes and Mycobacterium tuberculosis perform a 10-fold gradient dilution on recombinant standard plasmids pMD19-T-1mo0733 and pMD19-T-Rv2875 with known copy numbers, perform gPCR reactions with each dilution of the plasmid as template, with ddH:0 as negative control, and perform conventional PCR to analyze the sensitivity of qgPCR.

2. Fluorescent PCR method for detecting Listeria monocytogenes and Mycobacterium tuberculosis in milk according to claim 1, wherein in S3 the raw materials of the Lowenstein-Jensen culture medium consist of fresh egg fluid, 2% malachite green fluid, glycerol, magnesium sulfate, sodium glutamate , potassium dihydrogen phosphate, magnesium citrate and deionized water.