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Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof

A technology for detecting kits and animal chlamydia, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. It can solve problems such as multiple inspection methods for the diagnosis of swine chlamydia that have not yet been seen, and reduce the difficulty of design. , reduce the number of primers, improve the effect of sensitivity

Inactive Publication Date: 2009-12-23
CHONGQING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of pathogen detection at home and abroad, there have been many reports on the simultaneous detection of multiple pathogens using single-tube multiplex PCR, but in terms of veterinary clinical diagnosis, no diagnostic method for multiple inspection of Chlamydia swine has been seen.

Method used

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  • Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof
  • Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof
  • Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Embodiment 1, design and screening of primers of the present invention

[0117] Collect the full-length sequences of the ompA gene of 9 known subtypes of Chlamydia strains, use ClustW software to perform multiple alignments, and design specific primers for each subtype of Chlamydia in the specific region of each subtype of Chlamydia, which are respectively denoted as : SEQ ID NO1...SEQ ID NO6. All primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. Use the nucleic acid extraction kit to extract normal Hella cells, Chlamydia-free pig lung tissue and serum DNA, pre-denaturation: 95°C for 5 minutes; cycle: 94°C for 60s, 50°C for 60s, 72°C for 60s; 35 cycles, extension: 72°C 8min for amplification. Primers with significant amplification were removed. Use the nucleic acid extraction kit to extract the standard Chlamydia strains respectively, use the designed pairs of subtype-specific primers to amplify each positive Chlamydia nucleic acid template under t...

Embodiment 2

[0121] The preparation of embodiment 2 positive control substance

[0122] Use the nucleic acid extraction reagent of the kit to extract the DNA of the cell culture of positive Chlamydia strains, and electrophoresis the extracted nucleic acid. Then Mix I PCR was used to amplify the DNA of Chlamydia porcine, Chlamydia psittaci, and Chlamydia pneumoniae respectively, and a band of about 1100bp could be amplified after the three reactions. The amplified bands were recovered using a gel recovery kit, and the concentration of the recovered nucleic acid was measured using a spectrophotometer. Carry out ligation reaction with pMD19 vector at a ratio of 1:10, ligate at 23°C for 2 hours, transform JM109 bacteria, resistance screening and PCR identification positive, re-sequence verification, obtain a nearly full-length fragment clone containing the ompA gene, and three kinds of Chlamydia The positive plasmids were mixed in equal proportions, aliquoted to 50uL each, and the concentrati...

Embodiment 3

[0123] The preparation of embodiment 3 negative reference substance

[0124] Use the nucleic acid extraction reagent of the kit to extract the DNA of pig lung tissue without Chlamydia infection, and electrophoresis the extracted nucleic acid. Then dilute with sterilized deionized water, control the concentration at 80-100ng / uL, and dispense 50uL each.

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Abstract

The invention relates to a subtype diagnostic kit of animal chlamydia disease and a method thereof. In the invention, a pair of aiming upstream and downstream polymerase chain reaction oligonucleotide primers according to the three animal chlamydia ompA gene sequences of animal chlamydias, one specific fragment of all the animal chlamydias can be expanded, and the effective detection of all the chlamydias of animal bedsonia is realized. On the basis of the primers, three sections of fragments with different lengths and sizes can be synchronously expanded by a single pipe in a clinical nucleic acid specimen and can be clearly resolved in common electrophoresis so as to realize the synchronous detection of three animal chlamydias. The invention utilizes a sleeve type multiple PCR method to synchronously detect the three chlamydias of pig chlamydia, psittacosis chlamydia and pulmonitis chlamydia by the single pipe, thereby lowering the cost and the workload of the animal chlamydia disease diagnosis of an animal chlamydia laboratory. The invention has strong specificity, high sensitivity, high repeatability, simple and convenient operation and no complicated post-treatment.

Description

technical field [0001] The invention relates to an animal chlamydia quarantine detection kit, in particular to a multiple nested PCR for simultaneous identification of chlamydia (genus) and single-tube type detection of three common animal chlamydia, chlamydia porcine, chlamydia psittaci and chlamydia pneumoniae As for the detection kit, the present invention also uses the kit to carry out the biological detection method of synchronous multiple nested PCR detection on the three kinds of animal chlamydia. technical background [0002] Chlamydia suis, Chlamydophila psittaci, and Chlamydophila pneumoniae are three common chlamydiae that cause chlamydial disease in animals. In veterinary clinic, these three kinds of chlamydia are often mixed infection, causing abortion, diarrhea and respiratory symptoms in pigs. Laboratory infection experiments by Rogers et al. found that Chlamydia swine infection can cause asymptomatic intestinal damage without causing diarrhea (Rogers, D.G., ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 李应国李正国王昱王国民肖进文聂福平袁陵吴东海刘生峰谭志周启民
Owner CHONGQING UNIV