Triplex PCR detection primers and triplex PCR detection kit for cytolethal distending toxins A, B and C of Campylobacter

A detection kit and campylobacter detection technology, applied in the field of PCR detection, can solve the problems of inaccurate results, poor sensitivity, time-consuming and laborious, etc., and achieve the effects of reducing false positives, improving detection, and reducing the probability of occurrence

Active Publication Date: 2018-11-30
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it takes 2-7 days for bacterial isolation to be diagnosed, which is not only time-consuming and laborious, but also the result is inaccurate, and the sensitivity is poor, and fa...

Method used

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  • Triplex PCR detection primers and triplex PCR detection kit for cytolethal distending toxins A, B and C of Campylobacter
  • Triplex PCR detection primers and triplex PCR detection kit for cytolethal distending toxins A, B and C of Campylobacter
  • Triplex PCR detection primers and triplex PCR detection kit for cytolethal distending toxins A, B and C of Campylobacter

Examples

Experimental program
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Embodiment 1

[0054] Example 1, Triple PCR Detection Method of Campylobacter coli Cell Expansion Lethal Toxin Type A, Type B and Type C

[0055] (1) DNA extraction of Campylobacter coli strains

[0056] DNA extraction method can be conventional method, and the extraction method of DNA of the present invention is as follows:

[0057] Spread the sample to be tested on the solid medium, culture, scrape the colonies growing on the surface of the solid medium, and collect them into a clean centrifuge tube;

[0058] Add 1.5ml of lysis solution, the composition of the lysis solution is: 0.15mol / L of NaCl, 15-35 mmol / l of guanidine hydrochloride, 0.1mol / l of Na 2 EDTA, 15mg / ml lysozyme, 10-30 mmol / l dodecyl amino acid, pH=8.0;

[0059] 100°C water bath for 5 min;

[0060] Add 100 μl absolute ethanol, gently invert the centrifuge tube to mix evenly, and centrifuge;

[0061] Pour the solution and flocs in step ④ into the adsorption column of the DNA collection tube, and place at ro...

Embodiment 2

[0084] Example 2, Triple PCR Detection Method of Campylobacter jejuni Cell Expansion Lethal Toxin Type A, Type B and Type C

[0085] (1) DNA extraction of Campylobacter jejuni strains:

[0086] Method is with embodiment 1.

[0087] (2) Design primers

[0088] According to the gene of Campylobacter jejuni cell expansion lethal toxin type A, type B and type C, specific primers are designed, and the nucleotide sequence is as follows:

[0089] CJCDTAF: GCTACTTGGAATATGCAAGG (SEQ ID NO. 7)

[0090] CJCDTAR: AGGACTTGAACCTACTTTTC (SEQ ID NO. 8)

[0091] CJCDTBF: TGGTTTCCTATTRAAATCWCC (SEQ ID NO. 9)

[0092] CJCDTBR: AGGTGGAGTAGTTAAAAACC (SEQ ID NO. 10)

[0093]CJCDTCF: ATCTTTTAACCTTGCTTTTGC (SEQ ID NO. 11)

[0094] CJCDTCR: AAGGGGTAGCAGCTGTTAA (SEQ ID NO. 12)

[0095] Among them, w=a or t, r=g or a.

[0096] The above nucleotide sequences are in the direction from left to right of the 5'-end to the 3'-end.

[0097] The expected amplification lengths of Campylobacter jejuni ce...

Embodiment 3

[0105] Example 3, Triple PCR Detection Method of Campylobacter fetalis Cell Expansion Lethal Toxin Type A, Type B and Type C

[0106] (1) DNA extraction of Campylobacter fetalis strain:

[0107] Method is with embodiment 1.

[0108] (2) Design primers

[0109] According to the gene of Campylobacter fetal cell expansion lethal toxin type A, type B and type C, specific primers are designed, and the nucleotide sequence is as follows:

[0110] CFCDTAF: GTTTGGATTTTCAAATGTTCC (SEQ ID NO. 13)

[0111] CFCDTAR: AAGACAAATGTAAGCACTC (SEQ ID NO. 14)

[0112] CFCDTBF: TATTTATGCAAGTCGTGCGA (SEQ ID NO. 15)

[0113] CFCDTBR:AAGCATAAGTTTTGCAAACG (SEQ ID NO. 16)

[0114] CFCDTCF: CAAGAGTTCCTCTTAAACTC (SEQ ID NO. 17)

[0115] CFCDTCR: GGCTTTGCAAAACCAGAAG (SEQ ID NO. 18)

[0116] The above nucleotide sequences are in the direction from left to right of the 5'-end to the 3'-end.

[0117] The expected amplification lengths of Campylobacter fetalis cell expansion lethal toxin type A, type B...

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Abstract

The invention discloses triplex PCR detection primers and a triplex PCR detection kit for cytolethal distending toxins A, B and C of Campylobacter. The Campylobacter involved in the invention comprises Campylobacter coli, Campylobacter jejuni and Campylobacter fitus, and the nucleotide sequences of corresponding primers thereof are as shown in SEQ ID No. 1 to SEQ ID No. 18. A triplex PCR detectionmethod provided by the invention can quickly and accurately detect the cytolethal distending toxins of Campylobacter, and can realize detection of a single toxin or all the three toxins selected fromthe cytolethal distending toxins A, B and C of Campylobacter in one operation, so time and effort are saved, and the detection can be completed in on shot without three PCR operations and three electrophoresis operations; a conventional single PCR method usually takes 2-3 days, and the triplex PCR detection method of the invention shortens detection time to 4 h, so work efficiency is greatly improved; and the method can be used for general investigation of the toxin types of Campylobacter, molecular epidemiological investigation and vaccine screening and detection.

Description

technical field [0001] The invention relates to a triple PCR detection primer and a kit for Campylobacter cell expansion lethal toxin type A, type B and type C, and belongs to the technical field of PCR detection. Background technique [0002] The pathogenic name of campylobacter is solitary disease, which is a general term for various diseases of humans and animals caused by campylobacter. All kinds of animals (especially pigs, chickens, cattle, and sheep) can get sick. In addition to diarrhea, it can also cause abortion, infertility, mastitis, and infectious hepatitis in cattle, sheep, and dogs. In addition to causing diarrhea and food poisoning, it can also cause systemic infections such as miscarriage, sepsis, endocarditis, arthritis, pneumonia and meningitis. In recent years, there have been more and more reports on the isolation of Campylobacter from animals and humans at home and abroad. The disease is widely distributed around the world. At present, the disease ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/63
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143Y02A50/30
Inventor 李海利方剑玉张青娴朱文豪徐引弟王治方王克领郎利敏张立宪游一
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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