CN100554436C

CN100554436C - A kind of multiple PCR reaction kit and detection method thereof

Info

Publication number: CN100554436C
Application number: CNB200510011694XA
Authority: CN
Inventors: 吕占军; 李林
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-05-09
Filing date: 2005-05-09
Publication date: 2009-10-28
Anticipated expiration: 2025-05-09
Also published as: CN1861802A

Abstract

The present invention relates to biological technical field, disclose a kind of PCR diagnostic kit and detection method and application that is used to detect the milk cow foot rot.This PCR diagnostic kit detection milk cow foot rot has fast, high, the high specificity of susceptibility, low cost and other advantages.

Description

A kind of multiple PCR reaction kit and detection method thereof

Technical field

The present invention relates to biological technical field, specifically, relate to a kind of multiple PCR reaction kit and detection method thereof that detects the milk cow foot rot.

Background technology

Foot rot is one of principal disease of harm dairy, he mainly is the communicable disease that has that is caused by the mutual accompanying infection of multiple pathogenic bacteria, its sickness rate is higher, account for 30% of limb hoof disease, the generation of foot rot is all arranged in the diary farm of all parts of the world, and this seriously ill person can make milk cow not rise with crouching, and milk yield descends, mortality raises, and has brought very big financial loss to dairy.

Inspection to foot rot is tentatively to make a definite diagnosis by clinical symptom and pathological change at home, but ulcer at the bottom of dermatitis, the hoof between discriminating hoof inflammation, toe is had certain difficulty.Abroad ELISA method and PCR detection method have been used in the detection of foot rot, the ELISA test has very high specific, but its susceptibility is very low, is difficult for promoting.The PCR method has advantages such as quick, accurate, special, but just single pathogenic bacterium design is used at bacteroides nodosus, be under the bacteroides nodosus situation of which kind of serotype not isolating fully, also be not suitable for using.

Summary of the invention

The object of the present invention is to provide a kind of quick, high, high specificity of susceptibility that detects the milk cow foot rot, can realize suitability for industrialized production, the PCR diagnostic kit that cost is low and detection method and application.

The invention provides a kind of multiple PCR reaction kit, it is characterized in that it contains following primer:

(1) primer of detection necrobacillus:

Upstream primer 5 ' TGA CAT TAC AGC TGA AGC AAA 3 '

Downstream primer 5 ' TCC AAC AGA AAC AGA AGC ATC 3 '

(2) primer of detection Clostridium perfringens:

Upstream primer 5 ' TGT TAA GTT TGA GAC TTT TGC 3 '

Downstream primer 5 ' ATC TGG GAA TGC TGT ATA TTT 3 '

(3) primer of the golden staphylococcus of detection:

Upstream primer 5 ' CCG AAT TCG TTA TGA CAG AAT ACT 3 '

Downstream primer 5 ' CAA GTC GAC CAG CGT TGT CTT CGC 3 '

(4) primer of detection corynebacterium pyogenes:

Upstream primer 5 ' ACA ATT TGC GTT CGG ACA TCT 3 '

Downstream primer 5 ' CCA CTT GAG CCT GAA CTT TGC 3 '

Test kit of the present invention, wherein each component volume proportion is: sterilization ultrapure water 26 parts by volume, 10 * PCR reaction buffer, 5 parts by volume, 25mmol/L MgCl ₂4 parts by volume, 10mmol/L dNTP 2 parts by volume, 25pmol/L detect primer 1 parts by volume of necrobacillus, primer 1 parts by volume that 25pmol/L detects Clostridium perfringens, primer 1 parts by volume that 25pmol/L detects golden staphylococcus, primer 1 parts by volume that 25pmol/L detects corynebacterium pyogenes.

Preferably, test kit is formed 45 μ L reaction systems, the ultrapure water 26 μ L that wherein sterilize, 10 * PCR reaction buffer, 5 μ L, 25mmol/L MgCl ₂The primer 1 μ L of primer 1 μ L, the 25pm ol/L detection Clostridium perfringens of 4 μ L, 10mmol/L dNTP2 μ L, 25pmol/L detection necrobacillus, primer 1 μ L, the 25pmol/L that 25pmol/L detects golden staphylococcus detect the primer 1 μ L of corynebacterium pyogenes.

The present invention also provides the detection method with described test kit, and it comprises the steps:

(1) template DNA is added in the test kit, add the Taq archaeal dna polymerase rapidly, centrifugal;

(2) hatch on the pcr amplification instrument, the PCR loop parameter is:

94 ℃ of pre-sex change 5min;

94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations;

72 ℃ are extended 10min again;

(3) amplified production is carried out electrophoresis detection.

After the electrophoresis detection, the specific band of 404bp, 560bp, 668bp, 968bp appears wherein.

The invention also discloses the application of described test kit in detecting ox or pig necrobacillus, Clostridium perfringens, golden staphylococcus, corynebacterium pyogenes.

By detecting the result of necrobacillus, Clostridium perfringens, golden staphylococcus, corynebacterium pyogenes, come diagnosis of milk cow foot rot or diagnosis ox, pig necrobacillosis.

The present invention utilizes genetic engineering technique, the PCR reaction has high specificity, susceptibility advantages of higher, at the necrobacillus that is separated in the foot rot (Fusobacteriumnecrophorum), clostridium perfringens (Cl.Perfringense), gold-coloured staphylococci (Staphylococcus aureus) and corynebacterium pyogenes (Corynebacteriumpyogenes), these four kinds of bacterium can produce different toxin, specificity according to four kinds of verticillium toxin gene orders, design primer respectively, be combined into multiple PCR reaction kit after the optimization.With this test kit pathological material of disease or pathological material of disease are cultivated and to be increased the germ DNA that extracts behind the bacterium and carry out the PCR reaction, reaction product is through agarose gel electrophoresis, as three above specific bands, the existence that can make a definite diagnosis foot rot occur.

Product preparation process of the present invention is as follows:

(1) design primer

According to necrobacillus leukotoxin gene lktA gene is topmost virulence associated gene, with Oligo software this gene 404bp fragment is carried out design of primers;

In various toxin of clostridieum welchii and enzyme, the most important with alpha toxin, be main lethal toxin, with Oligo software the 560bp fragment of the Cpa gene of alpha toxin is carried out design of primers;

According to the nuC gene is the high conserved sequence of golden staphylococcus dna fragmentation, and this genes encoding gold staphylococcus TNase enzyme is that golden staphylococcus is peculiar, with Oligo software the 668bp fragment of nuC gene is carried out design of primers;

Very relevant according to bacillus pyogenes hemolysin pyolysin (PLO) with virulence, with Oligo software the gene 967bp fragment of this toxin of encoding is carried out design of primers.

Primer is synthetic by precious Tyke, Harbin biotech company.

(2) each carries out susceptibility and specificity test to primer by the PCR reaction pair;

(3) special primer to designed various bacterium is optimized combination;

(4) with behind four kinds of reference cultures extraction DNA, carry out PCR and react the primer of verifying combination, i.e. test kit.

(5) with laggard performing PCR reactions of extraction DNA such as condition bacterium intestinal bacteria, the checking test kit has specificity.

(6) gather pathological material of disease (or increase bacterium with pathological material of disease after), extract DNA, the practicality of checking test kit.

The preservation of diagnostic kit generally is kept in-20 ℃ the refrigerator, the short period of time planted agent time spent, can deposit in 4 ℃ of refrigerators.

Product advantage applies of the present invention exists:

1. this test kit has the susceptibility height when detecting the milk cow foot rot, and high specificity is diagnosed advantages such as quick.

2. this test kit production technique advanced person, production cost is low, the added value of product height, market application foreground is wide.

Embodiment

(1) gathers pathological material of disease

Gather pathological material of disease: prune ill hoof with scissors, moistening, the bad tissue of open position collecting part under hoof wall or the hoof that separates pulls to scrape with wooden disinfecting cotton swab of 2.5mm * 152mm or scalper and gets the diseased region pathological material of disease, answers carefulness to avoid the blood contamination sample.Pathological material of disease is inserted into and transports in the substratum.

Transport substratum: agar 0.4g halfcystine 0.05g methylene blue 0.02g Sodium phosphate dibasic 0.4g extractum carnis 1g adds water to 100ml, regulates pH value 7.2, heats 1210 ℃ of 15min, and is standby in the test tube of packing into.

(2) pathological material of disease is inoculated, increased number of bacteria

The preparation of substratum: hoof powder 4g, extractum carnis 0.5g, peptone 1g, NaCl 0.5g, yeast extract paste 0.1g agar 2g, add water to 100ml, regulating the pH value is 7.2-7.4, heats 121 ℃ of 15min.

The sample of gathering is inoculated on the substratum, cultivates 3～5d with anaerobic jar or anaerobism incubator 37 ℃ of following anaerobism.

(3) extraction of pathological material of disease pathogenic bacteria DNA,

DNA extraction method: according to gram negative bacillus DNA rapid extracting method.The pathological material of disease of in anaerobic culture medium, cultivating 37 ℃ cultivate 3～5d days after, picking colony, with the centrifugal again collection thalline of 800ulSTE suspension, suspend with 400ulTE, add 10ul N,O-Diacetylmuramidase (0.1mg/ml), hatched 15 minutes under 60 ℃, add 10ul Proteinase K (1% solution) and 5ulSDS (25%), to hatch under 60 ℃ 15 minutes. the cooling back adds equal-volume phenol on the ice cube, shakes centrifugal 10 minutes of 10000rpm/min.Supernatant liquor is moved on in another new EPPendorf pipe, add the equal-volume phenol/chloroform and shake centrifugal 10 minutes.Supernatant is moved on in another new EPPendorf pipe, add equal-volume chloroform/primary isoamyl alcohol, shake, centrifugal 10 minutes.Supernatant is moved on in another new EPPendorf pipe, and adding equal-volume 4M acetic acid is pressed the Virahol with 2 times of volumes, after fully shaking, and room temperature placement 10 minutes, centrifugal 10 minutes.Carefully remove supernatant liquor, obtain precipitation group, add 500ul70% ethanol, cleaned in centrifugal 10 minutes.More carefully remove supernatant liquor, drying at room temperature precipitation group, dissolution precipitation group in 50ulTE or in the aqua sterilisa.

The preservation of DNA: generally be kept in-20 ℃ the refrigerator.

(4) with the DNA that extracts pathological material of disease, add in the diagnostic kit, adding the Taq enzyme, in PCR reaction instrument, carry out PCR.

Test kit is formed:

45 μ L reaction systems: in Standard PC R pipe, sterilization ultrapure water 26 μ L, 10 * PCR reaction buffer, 5 μ L, 25mmol/L MgCl ₂4 μ L, 10mmol/L dNTP 2 μ L, four kinds of each 1 μ L (25pmol/L) of primer.

The PCR loop parameter:

94 ℃ of pre-sex change 5min at first; 94 ℃ of sex change 1min then, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 10min again.

Test kit is used:

After in the Standard PC R pipe in the template DNA 4.5 μ L adding test kit, add TaqDNA polysaccharase 0.5 μ L rapidly, hatch on the pcr amplification instrument by following loop parameter of short duration centrifugal back.(the Taq archaeal dna polymerase is the used enzyme of conventional biological test).After reaction finishes, take out the PCR pipe, amplified production is used for electrophoresis detection.

(5) use the PCR reaction product, at agarose gel electrophoresis, contrast Marker 2000 can differentiate the existence of foot rot or not have (Fig. 1).

Criterion: three bands occur or more than three bands; The necrobacillus specific band appears in all interior situations.

Description of drawings

Fig. 1 test kit detected result of the present invention figure.

Swimming lane 1: being the electrophorogram behind the PCR reaction product electrophoresis, from top to bottom is the electrophoresis band of corynebacterium pyogenes, golden staphylococcus, Clostridium perfringens, four kinds of bacterium of necrobacillus.

Swimming lane 2: be the standard substance Marker 2000 of contrast electrophoresis band.

Sequence table

＜110〉army of accounting for, Lu

Woods, Lee

＜120〉a kind of multiple PCR reaction kit and detection method thereof

<130>2005-05-09

<160>8

<170>PatentIn?version?3.3

<210>1

<211>21

<212>DNA

＜213〉artificial sequence

<400>1

tgacattaca?gctgaagcaa?a 21

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<400>2

tccaacagaa?acagaagcat?c 21

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<400>3

tgttaagttt?gagacttttg?c 21

<210>4

<211>21

<212>DNA

＜213〉artificial sequence

<400>4

atctgggaat?gctgtatatt?t 21

<210>5

<211>24

<212>DNA

＜213〉artificial sequence

<400>5

ccgaattcgt?tatgacagaa?tact 24

<210>6

<211>24

<212>DNA

＜213〉artificial sequence

<400>6

caagtcgacc?agcgttgtct?tcgc 24

<210>7

<211>21

<212>DNA

＜213〉artificial sequence

<400>7

acaatttgcg?ttcggacatc?t 21

<210>8

<211>21

<212>DNA

＜213〉artificial sequence

<400>8

ccacttgagc?ctgaactttg?c 21

Claims

1, a kind of multiple PCR reaction kit is characterized in that it contains following primer:

(1) primer of detection necrobacillus:

Upstream primer 5 ' TGA CAT TAC AGC TGA AGC AAA 3 '

Downstream primer 5 ' TCC AAC AGA AAC AGA AGC ATC 3 '

(2) primer of detection Clostridium perfringens:

Upstream primer 5 ' TGT TAA GTT TGA GAC TTT TGC 3 '

Downstream primer 5 ' ATC TGG GAA TGC TGT ATA TTT 3 '

(3) primer of the golden staphylococcus of detection:

Upstream primer 5 ' CCG AAT TCG TTA TGA CAG AAT ACT 3 '

Downstream primer 5 ' CAA GTC GAC CAG CGT TGT CTT CGC 3 '

(4) primer of detection corynebacterium pyogenes:

Upstream primer 5 ' ACA ATT TGC GTT CGG ACA TCT 3 '

Downstream primer 5 ' CCA CTT GAG CCT GAA CTT TGC 3 '.

2, test kit according to claim 1 is characterized in that, wherein each component volume proportion is: sterilization ultrapure water 26 parts by volume, 10 * PCR reaction buffer, 5 parts by volume, 25mmol/L MgCl ₂4 parts by volume, 10mmol/L dNTP 2 parts by volume, 25pmol/L detect primer 1 parts by volume of necrobacillus, primer 1 parts by volume that 25pmol/L detects Clostridium perfringens, primer 1 parts by volume that 25pmol/L detects golden staphylococcus, primer 1 parts by volume that 25pmol/L detects corynebacterium pyogenes.