CN102851390B - Preparation method of milk goat mastitis pathogen multi-PCR (polymerase chain reaction) detection kit - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and in particular relates to a preparation method of a multiple PCR detection kit for dairy goat mastitis pathogenic bacteria. The preparation method of a multiplex PCR detection kit for dairy goat mastitis pathogenic bacteria includes: 1) DNA extraction from samples to be tested; 2) setting up a PCR detection kit; 3) PCR amplification; 4) analysis of PCR amplification products. The invention has great significance for improving the detection and monitoring level of mastitis and ensuring food safety of goat milk and dairy products.

Description

Preparation method of multiplex PCR detection kit for dairy goat mastitis pathogen

1. Technical field:

The invention belongs to the technical field of microorganisms, and in particular relates to a preparation method of a multiple PCR detection kit for dairy goat mastitis pathogenic bacteria.

2. Background technology:

Goat milk and its dairy products have excellent nutritional value and huge economic benefits. In dairy goat breeding, an important source of goat milk, mastitis is a widespread infectious disease that reduces the production and quality of raw milk. Even cause serious economic losses. The main cause of mastitis is the infection of the breast by pathogenic bacteria. The main mastitis pathogenic bacteria are Staphylococcus aureus ( S. aureus ), Escherichia coli (E. coli), Streptococcus uberis ( Streptococcus uberis , S. uberis ), Streptococcus dysgalactiae , S. dysgalactiae , Streptococcus agalactiae , S. agalactiae , Klebsiella pneumonia, K. pneumonia . The detection and identification of mastitis pathogenic bacteria is not only an important basis for judging whether milk-producing dams suffer from mastitis, but also provides effective information for clinical prevention and treatment of mastitis. The traditional detection of pathogenic bacteria is based on bacterial isolation and biochemical identification experiments, which has a high false negative rate and is time-consuming.

3. Contents of the invention:

The purpose of the present invention is to provide a preparation method of a multiplex PCR detection kit for dairy goat mastitis pathogenic bacteria, which is of great significance for improving the detection and monitoring level of mastitis and ensuring the food safety of goat milk and its dairy products.

In order to achieve the above object, the technical solution adopted in the present invention is: a kind of milk goat mastitis pathogenic bacteria

The preparation method of heavy PCR detection kit is characterized in that: the preparation method is:

1) DNA extraction of samples to be tested;

2) Set up the PCR detection kit: composed of PCR reagent tubes, positive control, negative control, Taq DNA polymerase, bromophenol blue loading buffer (conventional use concentration);

3) PCR amplification: Add the pretreated DNA sample and Taq DNA polymerase into the PCR detection tube, and set the positive and negative controls at the same time, blow and mix with a micropipette, then centrifuge at high speed instantly, and perform the amplification reaction with a PCR machine;

4) Analysis of PCR amplification products: Perform gel electrophoresis on the above PCR amplification products, and compare the electrophoretic bands of the samples to be tested with the positive and negative controls to determine whether the samples to be tested are Staphylococcus aureus, Escherichia coli, Single or mixed infection of Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Klebsiella.

The reaction cycle conditions for PCR amplification reaction in step 3) are: pre-denaturation at 95°C for 4 min, denaturation at 94°C for 1 min, annealing at 60.5°C for 90 sec, extension at 72°C for 1 min, and 72°C after 30 cycles. After extending for 8 min, the PCR product was stored at 4°C for detection.

The multiplex PCR detection kit is based on the original PCR detection technology, and multiple single PCRs are placed in the same reaction system and under the same reaction conditions to achieve the detection of multiple pathogenic bacteria at the same time.

Compared with prior art, the advantages and effects that the present invention has are as follows:

1. A multiple PCR detection method for mastitis caused by single or mixed infection of dairy goat Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Klebsiella, Rapid, efficient and accurate detection of individual or mixed infection of Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Klebsiella pathogenic bacteria, This kit can also be used for molecular epidemiological investigation and curative effect monitoring of dairy goats infected with Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae and Klebsiella. The method is based on the knowledge of molecular biology, and avoids the interference of bacteria and impurity particles in the microscope examination of the traditional detection method, thereby greatly improving the diagnostic accuracy.

2. The present invention establishes a multiplex PCR rapid detection kit for 6 main pathogenic bacteria of mastitis, which can simultaneously detect multiple pathogenic bacteria, is a time-saving and efficient detection method, and can be used for clinical and subclinical The treatment of mastitis provides a scientific basis, and it is of great significance to improve the detection and monitoring level of mastitis, and to ensure the food safety of goat milk and its dairy products.

4. Description of drawings

Figure 1 is the single-plex PCR detection results of each strain (M: 100 bp DNA ladder; 1: Escherichia coli; 2: Streptococcus dysgalactiae; 3: Staphylococcus aureus; 4: Streptococcus agalactiae; 5: Breast Streptococcus; 6: Klebsiella; 7: negative control);

Figure 2 is a graph showing the results of multiplex PCR gel electrophoresis.

5. Specific implementation

The multiple PCR detection of the present invention is based on the original PCR detection technology, and multiple single PCRs are placed in the same reaction system and under the same reaction conditions, so that multiple pathogenic bacteria can be detected at the same time.

The selected multiplex PCR cloned genes were based on the fact that the Escherichia coli wecA gene belongs to the wec gene cluster, which encodes a transmembrane protein, the initial intestinal bacterial common antigen and the biosynthesis of O-antigen lipopolysaccharide. The nuc gene of Staphylococcus aureus encodes a heat-resistant nuclease specific for this strain, which has endonuclease activity, can degrade DNA and RNA, and can still maintain enzyme activity for 1 h at 100°C. Streptococcus agalactiae cbf gene encodes CAMP factor, which is the main virulence factor of Streptococcus agalactiae, which can cause CAMP phenomenon and reduce the function of host immune system. The pauA gene of Streptococcus uberis encodes PauA protein, which combines with plasminogen to form a plasminogen complex, and then stimulates the formation of blood fibrinolytic enzymes. It is one of the candidate antigens for studying streptococcal vaccines. The Klebsiella khe gene encodes the hemolysin protein, which can lyse red blood cells and is also one of the main virulence factors of Klebsiella. Compared with the 16s or 23s ribosomal gene, the 16S-23S spacer sequence faces less evolutionary selection pressure and a higher degree of variation, so it can be used for different species identification.

The present invention is mainly a multiplex PCR for simultaneously diagnosing and identifying clinical samples of Staphylococcus aureus, E. The method is realized through the following technical solutions:

1) DNA extraction from samples to be tested;

2) Set up the PCR detection kit: composed of PCR reagent tubes, positive control, negative control, Taq DNA polymerase, and bromophenol blue loading buffer (conventional use concentration);

3) PCR amplification: Add the pretreated DNA sample and Taq DNA polymerase into the PCR test tube, and set up positive and negative controls at the same time, blow and mix with a micropipette, then centrifuge at high speed for a moment, and perform the amplification reaction in a PCR machine. The cycle conditions of the PCR reaction were: pre-denaturation at 95°C for 4 min, denaturation at 94°C for 1 min, annealing at 60.5°C for 90 sec, extension at 72°C for 1 min, extension at 72°C for 8 min after 30 cycles, and preservation of the PCR product at 4°C for detection;

4) Analysis of PCR amplification products: Perform gel electrophoresis on the above PCR amplification products, and compare the electrophoretic bands of the samples to be tested with the positive and negative controls to determine whether the samples to be tested are Staphylococcus aureus, Escherichia coli, Single or mixed infection of Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Klebsiella;

The PCR detection tube in step 2) contains 10 × PCR buffer, deoxyribonucleic acid (dNTP), and 12 primers, of which 12 primers are artificially synthesized DNA fragments with the following base sequences (Table 1):

The positive control is a recombinant plasmid containing the target gene fragment. The construction method is to clone the expansion products into pMD18-T Simple Vector respectively, and then transform into TOP 10 competent cells. After PCR identification and sequencing, a positive recombinant plasmid is obtained. The plasmids were respectively Contains the corresponding target gene fragment; the negative control is sterilized double distilled water.

The advantage of the present invention is to provide a kind of milk goat Staphylococcus aureus, E. The PCR detection method can quickly, efficiently and accurately detect whether the individual to be tested is a single pathogenic bacteria of Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, and Klebsiella Or mixed infection, in addition, this kit can also be used for molecular epidemiological investigation and curative effect monitoring of dairy goats infected with Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Klebsiella . The method is based on the knowledge of molecular biology, and avoids the interference of bacteria and impurity particles in the microscope examination of the traditional detection method, thereby greatly improving the diagnostic accuracy.

Embodiment 1, the establishment of multiplex PCR reaction system

1. Primer design

Check the references and determine the undecylopentenyl phosphate-N-acetylglucosamine-1-phosphotransferase gene ( rfe for short) of escherichia coli, the thermostable nuclease leader gene ( nuc for short) of staphylococcus aureus, 16s-23s interval gene of Streptococcus dysgalactiae, CAMP factor gene ( cfb for short) of Streptococcus agalactiae, plasminogen activator gene ( pauA for short) of Streptococcus uberis, hemolysin gene of Klebsiella ( Khe for short) is the research object. Log in to the NCBI (www.ncbi.nlm.nih.gov) website, and download the nucleic acid sequences of the specific genes of the above-mentioned pathogens from the GeneBank database. According to the principles of primer design, multiplex PCR primers were designed using Primer Premier 5.0 software, as shown in Table 1.

2. Establishment of multiplex PCR reaction system

Firstly, carry out the single-plex PCR detection of each pathogenic bacteria respectively to determine the annealing temperature and the optimal reaction conditions; concentration) to determine the optimal conditions for the reaction.

3. Primer specificity and sensitivity detection

153 milk samples of clinical and subclinical mastitis dairy goats with single bacterial infection identified by traditional biochemistry were sampled. The specificity and sensitivity of the method were identified based on the above six pairs of multiplex PCR primers. The results are shown in Table 2.

Embodiment two, to the epidemiological investigation of 6 kinds of pathogenic bacteria of certain milk goat breeding base

1. Aseptically collect 100 milk samples from a dairy goat breeding base.

2. DNA extraction of the sample to be tested: Centrifuge the collected milk sample at 12 000 rpm × 5 min, discard the supernatant, collect the bacterial precipitate, add 1 × TE to wash once, suspend the bacterial cell with 50 μL 0.5 M NaOH, and pass through a boiling water bath After adding for 5 minutes, quickly cool in an ice bath, add 50 μL of 1 M pH 8.0 Tris-HCl buffer solution, mix thoroughly, and centrifuge at 12 000 rpm for 5 minutes. The supernatant is the DNA template and stored at -20°C;

3. Multiplex PCR detection kit, which includes:

1), PCR reagent tube:

Wherein, the above-mentioned PCR primers are DNA artificially synthesized fragments, which are diluted to 20 μM with ddH ₂ O for use.

2) Positive control: The control DNA template is a recombinant plasmid containing the target gene fragment.

3) Negative control: The control DNA template is plateau sterilized double distilled water.

4), Taq DNA polymerase

5), Gold View DNA dye

6) Bromophenol blue loading buffer

4. Multiplex PCR amplification: Add 2 μL of DNA template and 0.13 μL of Taq DNA polymerase to each PCR detection tube, and set positive and negative controls at the same time. The micropipette was pipetted and mixed, and then centrifuged at high speed for a short time, and the PCR instrument was used for the amplification reaction. The PCR reaction cycle conditions were as follows: pre-denaturation at 95°C for 4 min, denaturation at 94°C for 1 min, annealing at 60.5°C for 90 sec, extension at 72°C for 1 min, extension at 72°C for 8 min after 30 cycles, and PCR products were stored at 4°C for detection.

5. Analysis of amplified products: The amplified products were detected by gel electrophoresis, and the results are shown as follows:

SEQUENCE LISTING

the

<110> Northwest A&F University

the

<120> Preparation method of multiplex PCR detection kit for dairy goat mastitis pathogen

the

<130> 2012

the

<160> 6

the

<170> PatentIn version 3.3

the

<210> 1

<211> 50

<212> DNA

<213> wecA (rfe)

the

<400> 1

gtggttcgac gggcaaacca gcctccgacg gtacataatc gccaccatat 50

the

the

<210> 2

<211> 42

<212> DNA

<213> 16S-23S

the

<400> 2

tggaacacgt tagggtcgaa ctagaaaaac tcttgattat tc 42

the

the

<210> 3

<211> 51

<212> DNA

<213> nuc

the

<400> 3

tcacaaacag ataacggcgt aaatagcact tgcttcagga ccatatttct c 51

the

the

<210> 4

<211> 54

<212> DNA

<213> cfb

the

<400> 4

cgaccttttg gacaagtagt aagataccgg agttgtcact tgatcagcat gtac 54

the

the

<210> 5

<211> 52

<212> DNA

<213> pauA

the

<400> 5

cacaattatt aacacgagat cgacgtcagt aggatgagtg ataggttctt ca 52

the

the

<210> 6

<211> 40

<212> DNA

<213> khe

the

<400> 6

ggaagtgtgg ataaacggct ctcctgctcg gtgttattga 40

the

the

Claims (1)

1. a preparation method for the scorching pathogenic bacteria multiple PCR detection kit of milk goat breast, is characterized in that: described preparation method is:

1) sample DNA to be checked extracts;

2) PCR detection kit is set: by PCR Reagent Tube, positive control, negative control, Taq archaeal dna polymerase, tetrabromophenol sulfonphthalein sample-loading buffer, formed;

3) pcr amplification: add pretreated DNA sample and Taq archaeal dna polymerase in PCR detector tube, the positive, negative control are set simultaneously, micropipet piping and druming mixes rear moment high speed centrifugation, and PCR instrument carries out amplified reaction;

4) pcr amplification product analysis: above-mentioned pcr amplification product is carried out to gel electrophoresis, with the electrophoretic band that positive, negative control sample more to be checked occur, take and judge that whether sample to be checked is the single or polyinfection of streptococcus aureus, intestinal bacteria, streptococcus uberis, streptococcus dysgalactiae, streptococcus agalactiae, klebsiella;

The reaction cycle condition that PCR in described step 3) carries out amplified reaction is: 95 ℃ of denaturation 4 min, 94 ℃ of sex change 1 min, 60.5 ℃ of annealing 90 sec, 72 ℃ are extended 1 min, 30 rear 72 ℃ of extension 8 min of circulation, preserve PCR product to be checked in 4 ℃;

Described multiple PCR detection kit be take original PCR detection technique as basis, by a plurality of single PCR be placed in same reaction system, same reaction conditions carries out, and reaches the detection to various pathogens simultaneously; The primer of multiple PCR detection kit is:

A, bacterial classification: intestinal bacteria; Atopen: wecA; Up/down trip primer: gTGGTTCGACGGGCAAACCAGCCTC CGACGGTACATAATCGCCACCATAT;amplified fragments: 264;

B, bacterial classification: streptococcus dysgalactiae; Atopen: 16S – 23S transcribed spacer; Up/down trip primer: tGGAACACGTTAGGGTCG AACTAGAAAAACTCTTGATTATTC; Amplified fragments: 256;

C, bacterial classification: streptococcus aureus; Atopen: nuc; Up/down trip primer: tCACAAACAGATAACGGCGTAAATAG CACTTGCTTCAGGACCATATTTCTC; Amplified fragments: 235;

D, bacterial classification: streptococcus agalactiae; Atopen: cfb; Up/down trip primer: cGACCTTTTGGACAAGTAGTAAGATACC GGAGTTGTCACTTGATCAGCATGTAC; Amplified fragments: 199;

E, bacterial classification: streptococcus uberis; Atopen: pauA; Up/down trip primer: cACAATTATTAACACGAGATCGACG TCAGTAGGATGAGTGATAGGTTCTTCA; Amplified fragments: 151;

F, bacterial classification: klebsiella; Atopen: khe; Up/down trip primer: gGAAGTGTGGATAAACGGCT

cTCCTGCTCGGTGTTATTGA; Amplified fragments: 109.