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CN-120666057-B - Kit for detecting drug resistance of streptococcus suis penicillin and amoxicillin and application thereof

CN120666057BCN 120666057 BCN120666057 BCN 120666057BCN-120666057-B

Abstract

The invention provides a kit for detecting drug resistance of streptococcus suis penicillin and amoxicillin and application thereof, belonging to the technical field of molecular biological detection. The kit comprises a primer pair and AlwNI restriction enzyme, wherein the primer pair comprises primers PBP1a-2083F and PBP1a-2083R, and the sequences of the primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. The kit is used for detecting drug resistance of streptococcus suis penicillin and amoxicillin, has the advantages of high specificity, high sensitivity, low detection cost and low technical requirements, and provides a reliable technology for monitoring the drug resistance of clinical streptococcus suis penicillin and amoxicillin.

Inventors

  • WU ZONGFU
  • PENG ZEREN
  • ZHENG HAN
  • SUN JIE
  • PENG XIAOLI

Assignees

  • 南京农业大学

Dates

Publication Date
20260508
Application Date
20250702

Claims (6)

  1. 1. The application of the primer pair and the AlwNI restriction enzyme in preparing a kit for detecting drug resistance of streptococcus suis and amoxicillin is characterized in that the primer pair comprises primers PBP1a-2083F and PBP1a-2083R, sequences are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2, the primer pair is adopted for PCR amplification reaction, the PCR product is purified when a fragment with the size of 570bp appears, the purified PCR product is subjected to enzyme digestion reaction by the AlwNI restriction enzyme, the enzyme digestion product is subjected to agarose gel electrophoresis, and when only two strips with the size of 479bp and 91bp appear in a lane, the streptococcus suis to be detected is judged to be penicillin and amoxicillin drug resistant strains.
  2. 2. The method for detecting drug resistance of streptococcus suis penicillin and amoxicillin for the purpose of non-disease diagnosis is characterized by comprising the following steps: (1) Taking streptococcus suis bacterial liquid to be detected as a template, carrying out PCR amplification reaction by adopting the primers PBP1a-2083F and PBP1a-2083R in claim 1, carrying out agarose gel electrophoresis on the obtained PCR product, and purifying the PCR product if a fragment with the size of 570bp appears; (2) And (3) carrying out enzyme digestion reaction on the purified PCR product by adopting AlwNI restriction enzyme, carrying out agarose gel electrophoresis on the enzyme digestion product, and judging that the streptococcus suis to be detected is penicillin and amoxicillin resistant strain when only two bands with the sizes of 479bp and 91bp appear in a lane.
  3. 3. The method according to claim 2, wherein the PCR reaction system comprises 12.5. Mu.L of 2 XTaq Master mix, 8.5. Mu.L of ddH 2 O, 1. Mu.L of PBP1a-2083F, 1. Mu.L of PBP1a-2083R, and 2. Mu.L of bacterial liquid.
  4. 4. A method according to claim 3, wherein the PCR reaction is performed by a procedure of 95℃pre-denaturation for 5min, 95℃denaturation for 30 s,55℃primer annealing for 30 s,72℃extension for 30 s,30 cycles, and 72℃extension for 10 min.
  5. 5. The method according to claim 4, wherein the cleavage reaction is performed by 1. Mu.g of purified PCR product, 1. Mu.L of AlwNI enzyme, 5. Mu.L of 10X rCutSmart buffer, and ddH 2 O is used to make up the reaction to 50. Mu.L.
  6. 6. The method according to claim 5, wherein the cleavage reaction is carried out by heat-inactivating the endonuclease enzyme in a water bath at 75-85 ℃ for 15-25min after a water bath at 35-39 ℃ for 5-15 min.

Description

Kit for detecting drug resistance of streptococcus suis penicillin and amoxicillin and application thereof Technical Field The invention belongs to the technical field of molecular biology detection, and particularly relates to a kit for detecting drug resistance of streptococcus suis penicillin and amoxicillin and application thereof. Background Streptococcus suis (Streptococcus suis, SS) is an important pathogenic bacterium of pigs, is mainly colonized on the upper respiratory tract and tonsils of pigs, can cause meningitis, arthritis, pneumonia, septicemia and even death of pigs, and brings great economic loss to pig industry. Beta-lactam antibiotics, including penicillin and amoxicillin, are one of the main antibiotics for the treatment of swine streptococcosis. In recent years, streptococcus suis has developed resistance to beta-lactam antibiotics due to abuse of antibiotics. If the strain has generated penicillin and amoxicillin drug resistance, the continuous use can cause treatment failure, delay illness state and increase the death rate of sick pigs and the breeding cost. At present, the mechanism of drug resistance of streptococcus suis to beta-lactam antibiotics is not clear, the clinical detection of the drug resistance of streptococcus suis is mainly determined by measuring the Minimum Inhibitory Concentration (MIC) of antibiotics to bacteria, the time is 3-4 days, the technical requirement is high, and the method is not suitable for the rapid detection of a basic layer, so that a reliable and rapid method for detecting the drug resistance of the streptococcus suis to the penicillin and the amoxicillin is lacking in the prior art. Disclosure of Invention The invention aims to provide a kit for detecting drug resistance of streptococcus suis penicillin and amoxicillin, which has the advantages of high specificity, high sensitivity, low detection cost and low technical requirements, and provides a reliable technology for monitoring the drug resistance of clinical streptococcus suis penicillin and amoxicillin. The invention adopts the following technical scheme: the kit for detecting drug resistance of streptococcus suis penicillin and amoxicillin comprises a primer pair and AlwNI restriction enzyme, wherein the primer pair comprises primers PBP1a-2083F and PBP1a-2083R, and the sequences of the primers are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The method for detecting drug resistance of streptococcus suis penicillin and amoxicillin by adopting the kit for non-diagnosis purpose is characterized by comprising the following steps: (1) Taking streptococcus suis bacterial liquid to be detected as a template, adopting primers PBP1a-2083F and PBP1a-2083R to carry out PCR amplification reaction, carrying out agarose gel electrophoresis on the obtained PCR product, and purifying the PCR product if a fragment with the size of 570bp appears; (2) And (3) carrying out enzyme digestion reaction on the purified PCR product by adopting AlwNI restriction enzyme, carrying out agarose gel electrophoresis on the enzyme digestion product, and judging that the streptococcus suis to be detected is penicillin and amoxicillin resistant strain when two bands with the sizes of 479bp and 91bp appear in lanes. In the invention, the PCR reaction system is as follows, 12.5. Mu.L of 2X TAQ MASTER Mix, 8.5. Mu.L of ddH 2 O, 1. Mu.L of PBP1a-2083F, 1. Mu.L of PBP1a-2083R, 2. Mu.L of bacterial liquid. In the present invention, the PCR reaction procedure was as follows, 5min at 95℃for denaturation, 30 s at 95℃for primer annealing, 30 s at 55℃for extension, 30 s at 72℃for extension, and 10min at 72 ℃. In the present invention, the cleavage reaction system was 1. Mu.g of the purified PCR product, 1. Mu.L of AlwNI restriction enzyme, 5. Mu.L of 10X rCutSmart buffer, and ddH 2 O was used to make up the reaction system to 50. Mu.L. In the present invention, the procedure of the enzyme digestion reaction is as follows, the enzyme digestion reaction is performed by heat inactivation of the enzyme in a water bath of 75-85 ℃ for 15-25min after the enzyme digestion reaction is performed for 5-15min in a water bath of 35-39 ℃. The invention has the beneficial effects that the drug resistance detection of the streptococcus suis to penicillin and amoxicillin can be completed in 3-4 days by the conventional 96-hole polystyrene board micro-broth dilution method, and the drug resistance of the streptococcus suis to penicillin and amoxicillin can be detected in 4-5 hours by the kit. Therefore, in clinic, the kit can be used for preliminary detection, and although the drug-resistant strains of penicillin and amoxicillin can be missed, if a streptococcus suis strain is used for rapidly detecting the drug resistance to penicillin and amoxicillin, other antibiotic sensitivity experiments can be carried out as early as possible, so that antibiotics capable of being efficiently treated can be found, and pig farm losses can be reduced. The k