CN110564876A - Method for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs - Google Patents

Abstract

The invention provides a method for identifying whether type 1 riemerella anatipestifer is resistant to fluoroquinolone drugs and application thereof, belonging to the field of poultry pathology research.

Description

Method for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs

Technical Field

The invention belongs to the field of poultry pathology research, and particularly relates to a method for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs and application thereof.

Background

The riemerella anatipestifer type 1 can attack ducklings, goslings and young turkeys, and the characteristic pathological changes caused by the riemerella anatipestifer type 1 are perihepatitis, pericarditis, peritonitis and the like, which are commonly called infectious serositis. In 1932, the bacterial strain is discovered to be still popular all over the world and is one of the common bacterial diseases harming the aquatic bird culture in China. Fluoroquinolone drugs (norfloxacin, enrofloxacin, ciprofloxacin and the like) belong to third-generation quinolones, and are widely applied to treatment of poultry systemic infection due to the characteristics of wide antibacterial spectrum, high antibacterial activity, strong tissue penetrability and the like, and the clinical application amount of domestic veterinarians is second to that of beta lactam antibiotics. In recent years, the drug resistance of riemerella anatipestifer type 1 to fluoroquinolones is mostly reported, and how to rapidly determine the sensitivity of the bacterium to the fluoroquinolones and guide the selection of the drugs is very urgent. There are various mechanisms of resistance of bacteria to fluoroquinolones, and among them, mutation of DNA gyrase (target of action of fluoroquinolones) is one of important causes.

Earlier researches show that base groups 247-249 of quinolone drug resistance determining region (QRDR) of DNA gyrase A subunit gene of Riemerella anatipestifer type 1 are subjected to mutation from AGC to ATC, and the bacterium is ensured to be resistant to fluoroquinolone drugs. Based on the characteristics, the invention designs a pair of primers to cross the difference sites of QRDR, establishes a PCR method and amplifies the QRDR, and the PCR amplification products of the two types of bacteria (refer to the fluoroquinolone drug-resistant or drug-intolerant Riemerella anatipestifer type 1) are 771 bp. Due to the difference of the lengths of EcoRV enzyme restriction fragments in the nucleotide sequences in the amplification region, mutation from AGC to ATC results in addition of an EcoRV enzyme site (GAT ↓ ATC) in the region. That is, the PCR product of the bacterium of a fluoroquinolone drug-intolerant strain (here GATAGC) can be cleaved by EcoRV enzyme to a size-invariant (still 771 bp); the PCR product of the bacterial strain (here GAT ↓ ATC) resistant to fluoroquinolone drugs is cut into two different sections (244 bp and 527 bp) by EcoRV enzyme. The sensitivity of the method is equivalent to that of PCR, electrophoresis is carried out only after extra EcoRV enzyme digestion is carried out on a PCR product (glue recovery is not needed on the product), the method is simple and practical, drug sensitivity test is not needed, convenience and rapidness are achieved, a report of a method for identifying whether the riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs based on a similar principle is not found at present, and the research can fill the blank of related research fields.

Disclosure of Invention

The invention aims to provide a method for identifying whether the type 1 Riemerella anatipestifer is resistant to fluoroquinolone drugs, the sensitivity of the method is equivalent to that of PCR, and whether the type 1 Riemerella anatipestifer is resistant to the fluoroquinolone drugs can be identified according to an electrophoresis strip by only carrying out EcoRV enzyme digestion on a PCR product and then carrying out conventional agarose gel electrophoresis

In order to achieve the purpose, the invention adopts the following technical scheme:

A primer for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs or not is disclosed, and the primer sequence is as follows:

YF1：5’- ATGCATAAAGAAGGAGAAA-3’；

YR1：5’- AATAGCATTACGGTTTCCAA-3’。

A method for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs comprises the following steps:

(1) Extracting a strain genome;

(2) Amplifying a genome by PCR;

(3) And carrying out enzyme digestion identification on the PCR product by using EcoRV enzyme.

the invention has the beneficial effects that: the sensitivity of the method is equivalent to that of PCR, electrophoresis is carried out only after extra EcoRV enzyme digestion is carried out on a PCR product (glue recovery is not needed on the product), the method is simple and practical, drug sensitivity test is not needed, the method is convenient and quick, a report of a method for identifying whether the riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs based on a similar principle is not available at present, and the research can fill the blank of related research fields.

Drawings

FIG. 1 is an electrophoretogram of PCR amplification; wherein M: DL2000 molecular weight standard; 1: strain R28; 2: strain R93; 3: a DEV; 4: MDPV; 5: DuCV; 6: GPV; 7: and (5) sterilizing the deionized water.

FIG. 2 is a restriction enzyme map of the PCR amplification product; wherein M: DL2000 molecular weight standard; 1: strain R28; 2: strain R93.

FIG. 3 shows a paper sheet method drug sensitivity test.

Detailed Description

Example 1

1. Material

1.1 strains and strains

The experimental strain type 1 Riemerella anatipestifer R28 (the strain is not resistant to fluoroquinolones) and the Riemerella anatipestifer R93 (the strain is resistant to fluoroquinolones) are preserved by the animal husbandry and veterinary research institute of agricultural academy of sciences of Fujian province.

Control strains Duck Enteritis Virus (DEV), Muscovy Duck Parvovirus (MDPV), duck circovirus (DuCV) and duck Goose Parvovirus (GPV) for the test are preserved by animal husbandry and veterinary research institute of agricultural academy of agricultural sciences in Fujian province.

1.2 primer design

Nucleotide series analysis alignment was performed using Lasergene DNASAR according to the Riemerella anatipestifer DNA gyrase A subunit (DNA gyrase subunit A) gene of the National Center of Biotechnology Information, NCBI database, using primer design software Oligo 7.0, as follows:

YF1：5’- ATGCATAAAGAAGGAGAAA-3’，

YR1：5’- AATAGCATTACGGTTTCCAA-3’。

The primers were synthesized by Biotechnology engineering (Shanghai) Inc.

When the strain R28 and the strain R93 were amplified using YF 1/YR 1, the band sizes of the PCR products of the strain R28 and the strain R93 were 771 bp (they could not be distinguished by conventional agarose electrophoresis). However, the R28 strain and the R93 strain have characteristic differences of enzyme cutting sites of EcoRV enzyme (GAT ↓ ATC) in the amplification region, wherein the R93 strain has a GAT ↓ ATC sequence (which can be recognized by the EcoRV enzyme) in the amplification region, and the R28 strain does not (the general drug-resistant sequence is GATAGC, and no enzyme cutting recognition site of the EcoRV enzyme) in the amplification region.

1.3 Primary reagents

2 × TransTaq-T PCR SuperMix, EasyPure Viral DNA/RNA Kit, EasyPure bacterio Genomic DNA Kit, DNA molecular weight standards DL2000, FlyCut EcoRV enzymes were purchased from Beijing Quanjin Biotechnology Ltd.

2 establishment of test methods

2.1 extraction of genomic DNA

test control strains DEV, MDPV, DuCV and GPV corresponding genomic DNA was extracted according to the method of the easy pure Viral DNA/RNA Kit and frozen at-80 ℃ for later use.

The corresponding Genomic DNA was extracted from the R28 strain and the R93 strain according to the method of the EasyPure Bacteria Genomic DNA Kit and frozen at-80 ℃ for use.

2.2 configuration of reaction solution and optimization of annealing temperature

Amplification was performed according to the 50. mu.L system recommended by the 2 XTrTaq-T PCR Supermix kit, wherein 25. mu.L of the 2 XTrMaster Mix amplification reaction solution, 1.0. mu.L of the primer YF 1/YR 1 (10. mu.M), 1.0. mu.L of the extracted genomic DNA template, and 50. mu.L of sterile deionized water were supplemented to the final volume, and PCR amplification was performed after mixing.

The amplification conditions are pre-denaturation at 94 ℃ for 5 min and then circulation, denaturation at 94 ℃ for 30 s, annealing at delta T (52 ℃ -62 ℃) for 30 s and extension at 72 ℃ for 45s, final extension at 72 ℃ for 10 min after 30 cycles are finished, and identification is carried out according to conventional agarose gel electrophoresis after reaction is finished. Delta T (52 ℃ -62 ℃) indicates that the annealing temperature is optimized in the interval, and the optimized optimal annealing temperature is 56 ℃.

As a result, it was found (FIG. 1) that a band of 771 bp in size appeared when the template was added to only the R28 strain (lane 1); when the template was added to only the R93 strain, a band of 771 bp in size appeared (lane 2), which was not visually distinguishable by conventional agarose electrophoresis.

2.3 specificity test

The optimized PCR system and the amplification conditions are used for amplifying DEV, MDPV, DuCV, GPV and sterilized deionized water control, no amplification band is found, and the result is shown in figure 1, DEV (lane 3), MDPV (lane 4), DuCV (lane 5), GPV (lane 6) and sterilized deionized water (lane 7), which shows that the established method has strong specificity and has no cross reaction to common waterfowl pathogens.

2.4 enzyme digestion identification

carrying out enzyme digestion identification on the PCR product by using FlyCut EcoRV enzyme, wherein the enzyme digestion system is a 20 mu L system: of these, 10. mu.L FlyCut Buffer, 10. mu.L FlyCut EcoRV enzyme 2. mu. L, PCR product, was supplemented with sterile deionized water to a final volume of 20. mu.L. Mixing the mixture gently, centrifuging the mixture instantly, and reacting the mixture in water bath at 37 ℃ for 15 min. The results are shown in FIG. 2, wherein two products of the strain R93 are obtained, and the sizes of the two products are 244 bp and 527 bp respectively (lane 2); the size of the cleavage product of strain R28 remained 771 bp (lane 1). The bacterial strain type 1 Riemerella anatipestifer R28 is not resistant to fluoroquinolone medicaments, and the Riemerella anatipestifer R93 is resistant to fluoroquinolone medicaments.

2.5 bacterial susceptibility test

Performing drug sensitivity test of bacteria by using a paper diffusion method, and judging that the riemerella anatipestifer R28 is sensitive to norfloxacin, ciprofloxacin, ofloxacin and enrofloxacin with the diameters of inhibition zones of 19 mm, 24 mm and 26 mm respectively; the diameters of inhibition zones of riemerella anatipestifer R93 on norfloxacin, ciprofloxacin, ofloxacin and enrofloxacin are respectively 12 mm, 14 mm and 15mm, and the riemerella anatipestifer is judged to be drug-resistant. See fig. 3.

3 clinical applications

extracting corresponding nucleic acid DNA from 25 parts of clinically separated Riemerella anatipestifer type 1, and identifying whether the Riemerella anatipestifer is resistant to fluoroquinolone medicaments by using an optimized method. After PCR amplification is carried out on the fluoroquinolone derivatives by YF 1/YR 1, EcoRV enzyme digestion identification is carried out, and the result shows that 9 strains exist with the product size of 771 bp, which indicates that the 9 strains are all intolerant to fluoroquinolone drugs and account for 36.0 percent; the product size is found to be 16 fragments of 244 bp and 527 bp, which shows that the 16 fragments are resistant to fluoroquinolone drugs and account for 64.0 percent. The result of comparison with the drug sensitivity test in 2.5 shows that 16 strains identified by the method of the invention are resistant to fluoroquinolone drugs, and the drug sensitivity test is resistant to fluoroquinolone drugs, so that the coincidence rate is 100%; the 9 strains identified by the method of the invention are all non-drug resistant to fluoroquinolone drugs, and drug sensitivity tests show that the strains are all non-drug resistant to fluoroquinolone drugs, and the compliance rate is 100%.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province

<120> method for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs

<130> 2

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence

<400> 1

atgcataaag aaggagaaa 19

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

aatagcatta cggtttccaa 20

Claims (2)

1. A primer for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs is characterized in that the primer sequence is as follows:

YF1：5’- ATGCATAAAGAAGGAGAAA-3’；

YR1：5’- AATAGCATTACGGTTTCCAA-3’。

2. A method for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs is characterized by comprising the following steps: the method comprises the following steps:

(1) extracting a strain genome;

(2) Amplifying a genome by PCR;

(3) And carrying out enzyme digestion identification on the PCR product by using EcoRV enzyme.