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Pair of specific primers for detecting Bergeyella, kit and PCR detection method

A detection kit, Bergeyella technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of cumbersome, expensive, time-consuming, etc. Effect

Pending Publication Date: 2018-12-21
HENAN UNIV OF ANIMAL HUSBANDRY & ECONOMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the identification of Bergeia spp. is mainly through staining microscopy, biochemical tests, 16S rRNA gene sequencing and comparison, which is very tedious, time-consuming, and expensive. Therefore, it is urgent to establish a fast and simple diagnostic method

Method used

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  • Pair of specific primers for detecting Bergeyella, kit and PCR detection method
  • Pair of specific primers for detecting Bergeyella, kit and PCR detection method
  • Pair of specific primers for detecting Bergeyella, kit and PCR detection method

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Effect test

Embodiment 1

[0028] Embodiment 1, the establishment of the PCR detection method of Bergeyella

[0029] 1.1 Primer design

[0030]The present invention downloads Streptococcus, Erysipelas suis, Pasteurella multocida, Salmonella, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Stenotrophomonas maltophilia, and Bergeys suis from the GenBank database , the 16S rRNA gene sequence of Bergeia zoolcers, sequence comparison was carried out by MEGA5.05 software, and the specific sequence of 16S rRNA of Bergeia was found, and then the partial sequence of the 16S rRNA gene of Bergeia D658 bacterial strain in the GenBank database (login No. NR_104718.1) as a reference sequence, select the 170bp-201bp site sequence of the gene fragment as the upstream primer design selection sequence and the 419bp-450bp site sequence as the downstream primer design selection sequence, and use Primer-BLAST design to only amplify Berger Bacteria a pair of specific primers, specifically as follows:

[0031] Upstre...

Embodiment 2

[0044] Embodiment 2, detection method verification

[0045] Utilize the establishment of the Bergeys PCR detection method, 11 strains of Bergeys (its bacterial strains are respectively named as YLD7, SQT1, DC-10, DJ-5, DY-10, DY-8, SQT6, JS2-4, XCSF, ZWSF, FqWF2) were detected, wherein FqWF2 was Bergeia suis, and others were Bergeia zoolis. The results showed that the 11 strains of Bergeyella PCR amplified bands were all 266bp in size, that is, the amplification result was positive ( figure 1 ). It is further illustrated that the established Bergeia PCR detection method can detect isolated and preserved Bergeria.

Embodiment 3

[0046] Embodiment 3, specific detection

[0047] Using Bergeia zoonoticum and 10 common bacteria as detection samples, the established PCR detection method for Bergeia was used for specific detection. The 10 bacteria are Escherichia coli, Salmonella, Proteus, Haemophilus parasuis, Stenotrophomonas maltophilia, Pasteurella, Streptococcus, Erysipelas suis, Actinobacillus pleuropneumoniae, Aeromonas . The results showed that the established PCR detection method for Bergeria was positive for Bergeria animal ulcers, and the other 10 common bacteria were all negative ( figure 2 ). It is further explained that the established PCR detection method for Bergeyella has strong specificity, and only Bergeria is detected as positive, and other non-Bergeytes are detected as negative.

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Abstract

The invention discloses a pair of specific primers for detecting Bergeyella, a kit and a PCR detection method. The primers comprise an upstream primer Bergeyella-F: 5'-TTGAAAGCTCCGGCGGATAG-3' and a downstream primer Bergeyella-R: 5'-ACCCTCACGAGAGTAGGTTT-3'. According to the specific primers, the kit and the PCR detection method, streptococcus, bacillus erysipelatos-suis, pasteurella multocida, salmonella, haemophilus parasuis, infectious actinobacillus pleuropneumoniae, stenotrophomonas maltophilia, pig Bergeyella and a Bergeyella zoohelcum 16S rRNA gene sequence are downloaded from a GenBankdatabase; sequence comparison is carried out through MEGA 5.05 software to search a Bergeyella 16S rRNA specific sequence; and Primer-BLAST design is utilized to amplify a pair of specific primers ofBergeyella; and a Bergeyella PCR rapid detection method is established to identify Bergeyella. The method has the advantages of being rapid, convenient, high in sensitivity and strong in specificity.

Description

technical field [0001] The invention relates to a detection method for bacteria, in particular to a pair of specific primers, a kit and a PCR detection method for detecting Bergeria. Background technique [0002] The genus Bergeyella (Bergeyella) is a Gram-negative, non-spore-forming, oxidase-positive, non-fermenting bacterium that was proposed by Vandamme in 1994. Currently, the genus Bergeyella includes two species, Bergeyella zoohelcum and Bergeyella porcorum. [0003] B. zoonoticum is mainly found in the oral or nasal microbiota of cats, dogs, or other mammals. In 2002, A.DECOSTERE et al. reported for the first time that Bergeia ulcerans was related to feline respiratory disease. B. zoolcers may cause rare but severe human clinical lesions after bites by dogs or cats, such as cellulite, leg abscesses, sepsis, etc. [0004] In 2016, Spanish scholar L.Zamoraa et al. isolated 4 strains of rod-shaped bacteria that were Gram-negative and positive in contact enzyme and oxid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2565/125Y02A50/30
Inventor 蒋增海邓同炜徐耀辉吕玉金赵攀登彭志峰
Owner HENAN UNIV OF ANIMAL HUSBANDRY & ECONOMY
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