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Quantitative detection method of resistance genes of intestinal bacterial antibiotics

A quantitative detection method and technology of antibiotic resistance, applied in the field of quantitative detection of antibiotic resistance genes of intestinal bacteria, can solve the problems of underestimating the number of intestinal microorganisms and the deviation of the carrying amount of intestinal bacteria resistance genes, and achieve high theoretical research. Significance and practical value, stable and reliable results, accurate test results

Inactive Publication Date: 2017-10-17
SUN YAT SEN UNIV
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  • Description
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  • Application Information

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Problems solved by technology

Therefore, the traditional microbial culture method inevitably underestimates the number of intestinal microorganisms, and there is a large deviation in the evaluation of the carrying amount of intestinal bacterial resistance genes

Method used

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  • Quantitative detection method of resistance genes of intestinal bacterial antibiotics
  • Quantitative detection method of resistance genes of intestinal bacterial antibiotics
  • Quantitative detection method of resistance genes of intestinal bacterial antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Screening and identification of antibiotic-resistant strains

[0039] 1. Sample collection was carried out at Guanli Marine Biotechnology Co., Ltd., Maoming City. The specification of Litopenaeus vannamei is 50-60 tails / catties, the body length is about 13-15cm, and the weight is about 10g. After the prawns were temporarily raised in the aquarium for 2 days, they were randomly divided into 5 groups, with 80 prawns in each group. According to the weight of the shrimp body, different concentrations of antibiotics were added, as shown in Table 1. After the antibiotics were dissolved and mixed with the feed, they were fed into the aquarium, and the feed dosage was 1% of the body weight of the shrimp.

[0040] Table 1 Group design situation

[0041] Group

Types of

antibiotic type

Antibiotic Concentration

Cont

control group

Do not use

CipH

High concentration ciprofloxacin group

Ciprofloxacin

40mg / kg shrim...

Embodiment 2

[0063] Example 2 Real-time quantitative detection of intestinal bacterial resistance genes of Litopenaeus vannamei

[0064] 1. Using the method established in Example 1, carry out the antibiotic resistance gene before feeding antibiotics, the 1st day after feeding, the 2nd day after feeding, the 3rd day after feeding, and the 4th day after feeding Quantitative PCR detection.

[0065] Before administration of ciprofloxacin, intestinal qnr B copies / 16s copies is 3.37×10 -3 , qnr D copies / 16s copies are 5.34×10 -4 , qnr S copies / 16s copies is 3.30×10 -3 .

[0066] Before feeding sulfamethazine, sul 1 copies / 16s copies is 2.53×10 -3 , sul 2 copies / 16scopes is 2.21×10 -2 , sul 3 copies / 16s copies is 5.55×10 -4 .

[0067] 2. Changes in antibiotic resistance genes

[0068] (1) For qnr B gene, in the CipH group, qnr B copies / 16 copies were 6.73×10 from the first day to the fourth day after feeding -2 , 9.74×10 -2 , 1.15×10 -1 , 1.11×10 -1 . In the CipL group, ...

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Abstract

The invention discloses a quantitative detection method of resistance genes of intestinal bacterial antibiotics; the method comprises the steps of S1, extracting total DNA of intestinal bacteria; S2, using the total DNA of the intestinal bacteria as a template, amplifying antibiotic resistance genes and 16SrRNA gene via PCR (polymerase chain reaction), and linking the amplification products to cloning vectors, and converting host bacteria to obtain positive clones; propagating the positive clones, and extracting plasmids, measuring copy density of the plasmids, diluting at 100 gradient to obtain a standard template, and establishing a standard curve for the copy number and Ct value through fluorescent quantitative PCR; S3, subjecting the positive plasmids and samples to fluorescent quantitative PCR amplification, and calculating the copy number of antibiotic resistance genes carried in the samples to obtain relative content of the antibiotic resistance genes. The method is good in safety and environmental friendliness and low in price, has simple steps, is low in time consumption, has stable and reliable results, has good repeatability, high sensitivity and high practicality, is simple and fast, and has a promising application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a quantitative detection method for antibiotic resistance genes of intestinal bacteria. Background technique [0002] In recent years, due to the extensive use of antibiotics in aquaculture and animal husbandry, the problem of environmental pollution has become increasingly serious, and it has become one of the current international research hotspots. my country is a big country for Litopenaeus vannamei farming, but in recent years, due to the aggravation of aquaculture pollution, the quality of the aquaculture environment has continued to deteriorate, the variety of pathogenic organisms has increased and the speed of transmission has accelerated, and aquaculture diseases have become increasingly serious. The disease problems in aquaculture are especially bacterial diseases. It occurs frequently and seriously in intensive farming systems, which greatly limits the deve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12R1/01
CPCC12Q1/6851C12Q2531/113C12Q2563/107
Inventor 黄志坚曾燊正刘健侯冬伟何建国
Owner SUN YAT SEN UNIV
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