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Primer and probe combination and application for Chlamydia psittacosis genotype detection

A technology of Chlamydia psittaci, genotyping, applied in the field of molecular biology

Active Publication Date: 2020-07-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, no matter based on the PCR-RFLP method or the fluorescent quantitative PCR method, there is no mature kit for the genotype diagnosis of Chlamydia psittaci in the domestic market. Fewer, so far there is no mature solution for high-throughput and high-efficiency detection and analysis of C. psittaci genotypes by gene chip method

Method used

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  • Primer and probe combination and application for Chlamydia psittacosis genotype detection
  • Primer and probe combination and application for Chlamydia psittacosis genotype detection
  • Primer and probe combination and application for Chlamydia psittacosis genotype detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 The screening of Chlamydia psittaci genotype-specific probes and primers

[0072] 1. Materials

[0073] Chlamydia probes and primers: Sangon Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize them.

[0074] Gene chip spotting instrument, PersonalArrayer TM 16-person spotting instrument: provided by Beijing Boao Jingdian Biotechnology Co., Ltd.

[0075] Bacterial nucleic acid extraction kit: EasyPure Bacteria Genomic DNA Kit (EasyPure Bacteria Genomic DNA Kit, catalog number: EE161-01) from Quanshijin (Beijing) Biotechnology Co., Ltd.

[0076] Nylon Membranes (Nylon Membranes, positively charged, Roche Company): purchased from Sigma-Aldrich Company.

[0077] Horseradish peroxidase-labeled streptavidin (streptavidin-HRP): purchased from Thermo Fisher Scientific.

[0078] Gene chip supporting reagents (M1, M2, M3, W4) were purchased from Shanghai Jieyuan Biotechnology Co., Ltd. Prepare the chip working solution according to the ratio specif...

Embodiment 2

[0095] The preparation of embodiment 2 Chlamydia psittaci genotype-specific gene chip

[0096] In this example, a chip is prepared from the optimal primers and probes finally obtained as described in Example 1, and the hybridization conditions and color development conditions of the chip are optimized, and finally the best reaction system for making the chip is selected to ensure the stability of the chip. The test results are optimal.

[0097] 1. Materials

[0098] Chlamydia probes and primers: Sangon Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize them.

[0099] Gene chip spotting instrument, PersonalArrayerTM16 personal spotting instrument: provided by Beijing Boao Jingdian Biotechnology Co., Ltd.

[0100] Bacterial nucleic acid extraction kit: EasyPure Bacteria Genomic DNA Kit (EasyPure Bacteria Genomic DNA Kit, catalog number: EE161-01) from Quanshijin (Beijing) Biotechnology Co., Ltd.

[0101] Nylon Membranes (Nylon Membranes, positively charged, ...

Embodiment 3

[0131] The construction of embodiment 3 chlamydia psittaci plasmid standard reference substance

[0132] This embodiment establishes a set of matching plasmid standard reference products for the gene chip, thereby reducing the chance of purifying the standard genome from the chlamydia pathogen, reducing the chance of infection, and more conveniently verifying and detecting the efficiency of the gene chip; it is also used as a Chlamydia species-specific The standard reference product matched with the chip detection kit can establish a positive control in the process of clinical sample testing.

[0133] 1. Materials:

[0134] Standard strains of each genotype:

[0135] genotype Standard strain A VS1 B CP3 C GR9 D NJ1 E CPMN F VS225 E / B WS / TR / E30 M56 M56 WC WC

[0136] Standard strains or genomes of Chlamydia psittaci A, B, C and D genotypes have been preserved in our laboratory.

[0137] Bacterial Genomic ...

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Abstract

The invention provides a primer for detection of the genotype of chlamydia psittaci, a probe combination and application. According to the allelism and equivalence property between the serotype and ompA genotype of the chlamydia psittaci (C.psittaci), by analyzing and screening gene sequences of VD1-VD4 areas of the ompA genotype of the chlamydia psittaci, a specificity probe and primer of the C.psittaci genetype are designed. The invention further provides a genotype identification chip of an ompA gene specificity area manufactured by the probe. A novel micro-array chip method for quickly andeffectively detecting the C.psittaci genotype with high flux is good in sensibility and specificity, a novel practical and effective detection method is provided for diagnosis, analysis and researchon the genotype of the chlamydia psittaci, and an effective means is provided for diagnosis of the chlamydia psittaci, epidemiological investigation and prevention and control over infectious diseases.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to the combination and application of primers and probes for genotype detection of Chlamydia psittaci. Background technique [0002] Chlamydia psittaci (C.psittaci) is a strictly eukaryotic intracellular parasitic zoonotic pathogen, which was first isolated from parrots. As a natural focal pathogen, its host range is extremely wide , can cause infection of humans, poultry, wild poultry, livestock and cause various diseases. The transmission of C. psittaci mainly occurs in birds and poultry, causing severe respiratory diseases, digestive tract diseases and reduction in production performance of poultry. Because some of its infection status is recessive infection, it will cause long-term damage and loss to the poultry breeding industry. . When humans come into contact with animals infected with C. psittaci, they mainly inhale infected poultry feces, secretions, and infec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6837C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6837C12Q1/689C12Q2600/156
Inventor 何诚郭永霞王艺晖曲光刚沈志强
Owner CHINA AGRI UNIV
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