Taqman real-time fluorescence PCR detection method for detecting Borrelia burgdorferi nucleic acid

A technology of Borrelia burgdorferi and real-time fluorescence, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, and resistance to vector-borne diseases. It can solve the problems of long detection time and achieve short detection cycle and high stability. , strong consistency effect

Inactive Publication Date: 2017-12-15
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a threat to the breeding industry and the safety of livestock and poultry products. However, the current Borrelia burgdorferi detection time is long, so there is an urgent need for a rapid detection method for Borrelia burgdorferi for import and export quarantine

Method used

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  • Taqman real-time fluorescence PCR detection method for detecting Borrelia burgdorferi nucleic acid
  • Taqman real-time fluorescence PCR detection method for detecting Borrelia burgdorferi nucleic acid
  • Taqman real-time fluorescence PCR detection method for detecting Borrelia burgdorferi nucleic acid

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Embodiment 1

[0023] Example 1 Taqman real-time fluorescent PCR detection of Borrelia burgdorferi in sick birds

[0024] Collect the whole blood from the wing vein of sick birds, centrifuge at 1000r / min for 10min, take 200-500μL of the pale white suspension at the junction of serum and blood cells, and put it into a 1.5mL centrifuge tube. The collected matter in the centrifuge tube was centrifuged at 12000r / min for 10min, then the supernatant was discarded, and the precipitate was collected for nucleic acid extraction; at the same time, the standard strain culture of Borrelia burgdorferi was set up as a positive control; SPF chicken blood was used as a negative control. You can also choose to collect poultry small intestine, spleen, liver, kidney, heart, lung and other tissues and organs as test samples.

[0025] Nucleic acid extraction: use a DNA extraction kit (purchased from Qiagen) to extract DNA, see the kit instructions for specific operations.

[0026] Real-time fluorescent PCR ampl...

Embodiment 2

[0032] The specificity test of the Taqman real-time fluorescent PCR detection method of embodiment 2 Borrelia burgdorferi

[0033] Three pathogens with similar symptoms after infecting poultry: chlamydia, mycoplasma and Theileria, and 20 strains of known bacteria were used as test strains for specificity experiments.

[0034] Table 1 Specific test strains

[0035]

[0036]

[0037] For the extraction of nucleic acid, the pure culture of the tested pathogen was used to extract nucleic acid respectively as a template for Taqman real-time fluorescent PCR detection, and a nucleic acid solution was obtained for future use.

[0038] Configuration and amplification parameters of the real-time fluorescent PCR system:

[0039] Amplification system: each 25μL amplification system contains 2×PremixExTaq TM Buffer 12.5 μL, 10 μmol / L upstream primer and downstream primer 1 μL each, 10 μmol / L probe 2 μL, nucleic acid template solution 1 μL, ultrapure water to make up the volume to 2...

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Abstract

The invention discloses a Taqman real-time fluorescence PCR detection method for detecting Borrelia burgdorferi nucleic acid, belonging to the technical field of biological detection. The Taqman real-time fluorescence PCR detection method disclosed by the invention applies a pair of Borrelia burgdorferi specific primers (SEQ ID NO. 1 and SEQ ID NO. 2 ) and a fluorescently labeled probe (SEQ ID NO. 3) for real-time fluorescence PCR amplification and detection of a fluorescence signal of an amplification product, so as to determine whether a sample carries Borrelia burgdorferi pathogen. The detection method is easy, rapid, sensitive, accurate and specific. The fluorescence probe which is complementary to and paired with a template enhances the specificity and effectively prevents false positives and false negatives. During detection, the fluorescence signals are collected automatically to avoid human factor interference in electrophoretic analysis after common PCR reaction, and a detection process is completely closed, without post-treatment such as electrophoresis, thereby eliminating the pollution of PCR products. The detection method disclosed by the invention has the characteristics of high specificity and good stability.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a Taqman real-time fluorescent PCR detection method for detecting Borrelia burgdorferi nucleic acid. Background technique [0002] Borrelia burgdorferi belongs to the Kingdom Monera, the order Spirochaetidae, the family Spirochaetaceae, and the genus Borrelia (also known as the genus Borrelia). Gram staining is negative, but it is not easy to stain. Giemsa staining and fluorescent dye staining are good, and elongated spirochetes can be seen under the microscope. Borrelia burgdorferi has multiple flagella, 3-10 large and sparse helices, 10-30 μm long and 0.2-0.25 μm wide, and consists of four parts: surface layer, outer membrane, flagella and protoplasm. In liquid medium, several spirochetes can be wound together irregularly and move in a twisted state. Transmitted by ticks, it can infect birds and other animals and cause Avian Spirochaetosis (AS). There is a threat ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2561/101Y02A50/30
Inventor 刘生峰陆丽华周庆杨俊张雷李贤良王国民聂福平王昱
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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