CN101984075A - Primer and kit for multiplex PCR detection of riemerella anatipestifer infection and colibacillosis and detection method thereof - Google Patents

Abstract

The invention relates to the technical field for diagnosis of poultry bacterial diseases, in particular to a primer set and a kit for multiplex PCR detection of riemerella anatipestifer and escherichia coli and a detection method thereof. The kit comprises 7 freezing tubes, wherein, 2 freezing tubes are respectively filled with 2*PCR core reagent premix, and the other 5 freezing tubes are respectively filled with a riemerella anatipestifer DNA solution, a riemerella anatipestifer positive DNA solution, a DL2000 DNA molecular weight standard solution, ddH2O and a primer mixture used in a PCR system. The detection method of the kit comprises the following steps: 1. providing DNA of a sample to be detected; and 2. taking the kit, amplifying the DNA of the sample to be detected by a conventional PCR method, detecting the amplification result by an agarose gel electrophoresis method, and finally judging according to the result. The multiplex PCR detection method established in the invention is specific, sensitive, simple, convenient and fast and can be applied to rapid detection of clinical samples and molecular epidemiological investigation and research of the riemerella anatipestifer and the escherichia coli.

Description

Riemerella anatipestifer sick and the detection of colibacillosis multiplex PCR primer and test kit and detection method thereof

Technical field

The present invention relates to the diagnostic techniques field of poultry bacterial disease, relate in particular to and be used for riemerella anatipestifer, bacillus coli multiple PCR and detect with primer sets test kit and detection method thereof.

Background technology

The riemerella anatipestifer disease is that (Riemerellaanatipestifer, acute or chronic, the contagious disease that RA) cause are mainly encroached on multiple birds such as duckling, goose by riemerella anatipestifer.This disease send out well in 2～7 age in week duck, the clinical nervous symptoms such as lassitude, appetite decline, cough and bend neck, limping that mainly show as are anti-ly crossed duck and are become stiff duck, growth retardation, its characteristics of lesion is fibrinous pericarditis, serohepatitis, airsacculitis etc.This disease sickness rate can reach more than 20%, and mortality ratio is up to 25%～57%, and this disease causes great financial loss to aviculture, is that one of the most serious bacterial infectious disease of duck industry is supported in influence.(Escherichiacoli E.coli) can cause multiple harm to various age in days ducks to the duck intestinal bacteria, and the most serious with 2～6 morbidities in age in week, sickness rate reaches 17%～56%, and the duck field mortality ratio that has is up to 43%.Two kinds of frequent polyinfections of disease; and all there are many similar places in the two at aspects such as clinical symptom, pathological change and epidemiology, and other two bacterium serotypes are numerous, and conventional diagnostic method is difficult to make quickly and accurately diagnosis; and cause mistaken diagnosis easily, affect treatment of diseases adversely.Therefore the PCR detection method of setting up a kind of differential diagnosis riemerella anatipestifer disease, colibacillosis and two kinds of disease polyinfections is very necessary.

Summary of the invention

In order to overcome the very difficult deficiency of making diagnosis quickly and accurately of prior art, first purpose of the present invention provides a kind of riemerella anatipestifer sick and colibacillosis multiplex PCR detection primer sets, second purpose of the present invention provides a kind of riemerella anatipestifer sick and colibacillosis multiple PCR detection kit, and the 3rd purpose of the present invention provides a kind of riemerella anatipestifer sick and colibacillosis multi-PCR detection method.That detection method of the present invention has is simple to operate, quick, specificity is good, the susceptibility advantages of higher.

In order to realize first above-mentioned purpose, the present invention has adopted following technical scheme.

Riemerella anatipestifer disease and colibacillosis multiplex PCR detect uses primer sets, and to constituting, two pairs of primers are to being respectively by two pairs of primers for this primer sets:

Silent Salmonella primer in the pest of duck:

5’-CCGTAGATTGGCTAACTTTTG-3’

5’-ATTTTAACTGAGATGGGT-3’；

The intestinal bacteria primer:

5’-TTCGTCGTTCTCATCCACAG-3’

5’-ACGCATAATAAACCCCATAG-3’。

In order to realize second above-mentioned purpose, the present invention has adopted following technical scheme.

Sick and the colibacillosis multiple PCR detection kit of riemerella anatipestifer, this test kit comprises following component: 2 * PCR core reagent pre-composition, silent Salmonella and intestinal bacteria positive control dna in the pest of duck, DL2000 molecular weight be with reference to solution, ddH2O and above-mentioned primer sets.

In order to realize the 3rd above-mentioned purpose, the present invention has adopted following technical scheme.

Sick and the colibacillosis multi-PCR detection method of riemerella anatipestifer, this method comprises the steps:

(1) provides testing sample DNA;

(2) adopt rabbit pasteurellosis bacillus as claimed in claim 2 and bordetella bacilli multiplex PCR to detect and use test kit, the DNA of multiple PCR method amplification testing sample adopts agarose gel electrophoresis method to detect amplification, judges according to the result; Described judge according to the result be specially, with the positive DNA of silent Salmonella and intestinal bacteria in the pest of duck in contrast,, then detect again if contrast does not amplify following whole band; If contrast amplifies following whole band, then the result judges as follows:

If the amplified fragments size is 670bp, be the silent Salmonella positive in the pest of duck;

If the amplified fragments size is 408bp, be the intestinal bacteria positive;

If increase 670bp, 408bp simultaneously, then be silent Salmonella and intestinal bacteria polyinfection in the pest of duck.

Outer membrane protein A (ompA) be riemerella anatipestifer and each serotype of intestinal bacteria total separately albumen, and verified this albumen conservative property in the various bacterial strain in various places is quite high, the present invention is a goal gene with riemerella anatipestifer and colibacillary ompA gene order, designed Auele Specific Primer, set up silent Salmonella in the rapid differential diagnosis pest of duck, the multi-PCR detection method of intestinal bacteria and polyinfection thereof, this method can directly detect pathogenic bacteria from the pathological material of disease tissue, do not need complicated DNA extraction step in addition, have simple to operate, fast, specificity is good, the susceptibility advantages of higher.Can be used for the rapid differential diagnosis and the epidemiology survey of riemerella anatipestifer and coli-infection, have a extensive future in public health, food safety, animal and veterinary and import and export inspection and quarantine or the like field.

The multiple PCR method that the present invention sets up does not need to increase bacterium and pure culture to test sample, can directly detect with pathological material of disease, has saved experimentation cost; Qin Zonghua etc. (2008) need increase bacterium and pure culture and complicated steps such as DNA extraction based on silent Salmonella disease and colibacillosis differential diagnosis dual-PCR method in the pest of duck of 16SrRNA foundation, the time of differentiating is longer, and the dual-PCR method that this test is set up is a template with bacterium liquid directly, from receiving pathological material of disease to detecting the result as long as 3～4h, shortened detection time, made it have practical value clinically.

Compared with prior art, beneficial effect of the present invention is as follows.

1, this multiple PCR method is simple to operate, has good specificity, and the different strains of bacterium of the same race is had extensive applicability.

2, have higher sensitivity, can detect that the silent minimum template detected level of Salmonella is 4 * 103cfu in the template pest of duck of trace level, the minimum template detected level of intestinal bacteria is 3 * 103cfu.

3, this multiplex PCR simultaneously in the pest of duck silent Salmonella and intestinal bacteria carry out differential diagnosis, compare quicker, economical with single PCR.

Description of drawings

Fig. 1 is multiplex PCR sensitivity Detection figure.M:DNA molecular mass standard; 1～7 (A, B) represents that respectively different dilution RA and E.coli bacterium liquid are the double PCR amplification of template, schemes wherein that bacterial count is respectively among the A: 1,8 * 10 ⁶, 8 * 10 ⁵, 8 * 10 ⁴, 8 * 10 ³, 8 * 10 ², 8,0.8, distilled water; Bacterial count is respectively among the figure B: 1,6 * 10 ⁶, 6 * 10 ⁵,, 6 * 10 ⁴, 6 * 10 ³, 6 * 10 ², 6,0.6, distilled water.

Fig. 2 is multiplex PCR specific detection figure as a result.M:DNA molecular mass standard; 1, RA ₁And E.coliO ₇₈Mixed culture; 2, RA ₁3, RA ₂4, RA ₁₀5, RA ₁₁6, E.coliO ₁7, E.coliO ₂8, E.coliO ₇₈9, E.coliO ₁₃₃10, duck source property pasteurella multocida; 11, duck source property Salmonellas; 12, distilled water.

Fig. 3 is multiplex PCR clinical detection figure as a result.M:DNA molecular mass standard; The clinical sample separation that 1～23:23 part is different; 24, negative control: distilled water.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

Embodiment 1

Test kit and detection method that present embodiment relates to are as follows:

1, test kit

The multiple PCR detection kit of present embodiment comprises 7 frozen pipes, 50 PCR reaction tubess and specification sheets.7 frozen pipes are respectively: the frozen pipe A(2 of code name) 2 * PCR core reagent pre-composition (Taq enzyme, dNTPs mixture and MgCl be housed ₂Solution, commercial), frozen pipe B and C are equipped with silent Salmonella and e. coli dna solution in the 50ul pest of duck respectively, and frozen pipe D is equipped with 0.5mlDL2000 molecular weight standard solution (commercial), and frozen pipe E is equipped with 1.5mlddH ₂O, frozen pipe F are equipped with PCR system the primer mixture, silent Salmonella primer: 5 '-CCGTAGATTGGCTAACTTTTG-3 ', 5 '-ATTTTAACTGAGATGGGT-3 ' in the pest of duck; Intestinal bacteria primer: 5 '-TTCGTCGTTCTCATCCACAG-3 ', 5 '-ACGCATAATAAACCCCATAG-3 '; All frozen Guan Jun are stored in-20 ℃.

2, the detection method of test kit

Step 1 is got frozen pipe A solution 12.5ul and is added respectively in the PCR reaction tubes, adds primer solution 2ul, carry out mark after, add the testing sample dna profiling immediately, with the ddH among the frozen pipe E ₂O transfers final volume to 25ul, detects after mixing; The positive control same operation adds silent Salmonella and positive each 2ul of dna solution of intestinal bacteria in frozen pipe B and the C pest of duck; Negative control does not add template, dH ₂O transfers final volume to 25ul.

Step 2 is placed on the PCR reaction tubes on the PCR instrument and increases: 95 ℃ of 5min; 94 ℃ of 40s, 52 ℃ of 40s, 72 ℃ of 45s, 35 circulations; 72 ℃ are extended 10min.

Step 3 is carried out 1.5% agarose gel electrophoresis with the product after the amplification under 110v voltage, the point sample amount of DL2000 molecular weight standard solution is the every hole of 8ul among PCR product and the frozen pipe D, and electrophoresis time 30min uses gel imaging analysis after electrophoresis finishes.

Step 4, the result judges:

(1) if silent Salmonella amplified fragments size is 670bp in the positive control pest of duck, the positive amplified fragments size of intestinal bacteria is 408bp, and the negative control result is negative, and then multiplex PCR system result judges as follows:

If testing sample amplifies the 670bp band, then be silent Salmonella in the pest of duck;

If testing sample amplifies the 408bp band, then be intestinal bacteria;

Do not have corresponding band or stripe size and misfit, then judged result is negative;

(2) if positive control does not amplify whole bands or positive purpose band appears in negative control, need detect again and judge.

Embodiment 2

The material that present case relates to is as follows: riemerella anatipestifer, intestinal bacteria, duck source property pasteurella multocida, duck source property Salmonellas reference strain are all available from China Veterinary Drugs Supervisory Inst..

Above-mentioned bacterial classification inoculation is increased bacterial context soup substratum in 2ml, and 37 ℃ of overnight incubation are carried out following test then.

1, sensitivity test

Riemerella anatipestifer, Escherichia coli bacteria liquid that 37 ℃ of shaking tables are spent the night carry out 10 times of serial dilutions, carry out carrying out multi-PRC reaction with detection sensitivity behind the plate count.Multiplex PCR sensitivity Detection result as shown in Figure 1, M:DNA molecular mass standard among Fig. 1; 1～7 (A, B) represents that respectively different dilution RA and E.coli bacterium liquid are the double PCR amplification of template, schemes wherein that bacterial count is respectively among the A: 1,8 * 10 ⁶, 8 * 10 ⁵, 8 * 10 ⁴, 8 * 10 ³, 8 * 10 ², 8,0.8, distilled water; Bacterial count is respectively among the figure B: 1,6 * 10 ⁶, 6 * 10 ⁵,, 6 * 10 ⁴, 6 * 10 ³, 6 * 10 ², 6,0.6, distilled water.

2, specificity test

Riemerella anatipestifer ( serum 1,2,10,11 types, abbreviate RA1 respectively as, RA2, RA10, RA11), (serum O1, O2, O78, O133 type abbreviate E.coliO1 respectively as to intestinal bacteria, E.coliO2, E.coliO78, E.coliO133), after recoveries such as duck source property pasteurella multocida, duck source property Salmonellas cultivate, extract DNA and carry out the specificity test.Multiplex PCR specific detection result as shown in Figure 2, M:DNA molecular mass standard among Fig. 2; 1, RA ₁And E.coliO ₇₈Mixed culture; 2, RA ₁3, RA ₂4, RA ₁₀5, RA ₁₁6, E.coliO ₁7, E.coliO ₂8, E.coliO ₇₈9, E.coliO ₁₃₃10, duck source property pasteurella multocida; 11, duck source property Salmonellas; 12, distilled water.

Test example 1

To from Hangzhou, Zhejiang province, Huzhou, Jiaxing, Shaoxing, Jinhua etc. from different places the duck that dies of illness of the appearance suspected case (cut open cubing pathologies such as pericarditis, serohepatitis and airsacculitis are arranged) of duck field censorship amount to 23 parts.Aseptic clip part liver organization is weighed, and adds PBS by 10 times of amounts (the 1g liver organization adds 10mLPBS) and grinds the back and use filter paper filtering, gets 2uL worry liquid and makes template and carry out pcr amplification according to the method for embodiment 2.

Cut open liver that inspection changes the duck that dies of illness with obvious serohepatitis, pericarditis and airsacculitis to 23 parts and carry out that bacterium separates and the PCR evaluation, the qualification result of 23 duplicate samples bacterium isolation identification results and PCR is consistent as a result, has further proved the reliability of this dual PCR detection method.The detected result of 23 duplicate samples shows have 6 duplicate samples to detect riemerella anatipestifer and intestinal bacteria polyinfection, accounts for 26.09% of institute's sample product; 12 duplicate samples and 5 duplicate samples are respectively riemerella anatipestifer and intestinal bacteria infect separately account for 52.17% and 21.74% of institute's sample product respectively; The detected result positive rate of riemerella anatipestifer and coli-infection is respectively 78.26% and 47.83%.

Multiplex PCR clinical detection result as shown in Figure 3, M:DNA molecular mass standard among Fig. 3; The clinical sample separation that 1～23:23 part is different; 24, negative control: distilled water.

Sequence table

＜110〉Zhejiang Academy of Agricultural Science

＜120〉sick and the detection of colibacillosis multiplex PCR primer and test kit and detection method thereof of riemerella anatipestifer

<160>4

 

<210>1

<211>21

<212>DNA

＜213〉synthetic

＜223〉primer

<400>1

CCGTAGATTGGCTAACTTTTG?21

 

<210>2

<211>18

<212>DNA

＜213〉synthetic

＜223〉primer

<400>2

ATTTTAACTGAGATGGGT?18

 

<210>3

<211>18

<212>DNA

＜213〉synthetic

＜223〉primer

<400>3

TTCGTCGTTCTCATCCACAG?20

 

<210>4

<211>18

<212>DNA

＜213〉synthetic

＜223〉primer

<400>4

ACGCATAATAAACCCCATAG?20

 

Claims (4)

1. primer sets is used in the sick and colibacillosis multiplex PCR detection of riemerella anatipestifer, it is characterized in that this primer sets by two pairs of primers to constituting, two pairs of primers are to being respectively:

Silent Salmonella primer in the pest of duck:

5’-CCGTAGATTGGCTAACTTTTG-?3’

5’-ATTTTAACTGAGATGGGT-?3’；

The intestinal bacteria primer:

5’-TTCGTCGTTCTCATCCACAG-?3’

5’-ACGCATAATAAACCCCATAG-?3’。

2. the sick and colibacillosis multiple PCR detection kit of riemerella anatipestifer, it is characterized in that this test kit comprises following component: 2 * PCR core reagent pre-composition, silent Salmonella and intestinal bacteria positive control dna in the pest of duck, the DL2000 molecular weight is with reference to solution, ddH2O and primer sets as claimed in claim 1.

3. the sick and colibacillosis multi-PCR detection method of riemerella anatipestifer is characterized in that this method comprises the steps:

(1) provides testing sample DNA;

(2) adopt rabbit pasteurellosis bacillus as claimed in claim 2 and bordetella bacilli multiplex PCR to detect and use test kit, the DNA of multiple PCR method amplification testing sample adopts agarose gel electrophoresis method to detect amplification, judges according to the result; Described judge according to the result be specially, with the positive DNA of silent Salmonella and intestinal bacteria in the pest of duck in contrast,, then detect again if contrast does not amplify following whole band; If contrast amplifies following whole band, then the result judges as follows:

If the amplified fragments size is 670bp, be the silent Salmonella positive in the pest of duck;

If the amplified fragments size is 408bp, be the intestinal bacteria positive;

If increase 670bp, 408bp simultaneously, then be silent Salmonella and intestinal bacteria polyinfection in the pest of duck.

4. the sick and colibacillosis multi-PCR detection method of riemerella anatipestifer according to claim 3 is characterized in that the pcr amplification parameter is specially: 95 ℃ of 5 min; 94 ℃ of 40 s, 52 ℃ of 40 s, 72 ℃ of 45 s, 35 circulations; 72 ℃ of 10 min.

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