Borrelia burgdorferi detection kit and application thereof

A Borrelia burgdorferi detection kit technology, applied in the fields of molecular biology and immunology, can solve the problems of single detection, lack of specificity, troublesome operation, etc., and achieve the effect of rapid detection and high sensitivity

Inactive Publication Date: 2018-12-28
吉林出入境检验检疫局检验检疫技术中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the results of etiological detection are reliable, it is cumbersome to operate and requires the operator's high skills and years of clinical experience to rely on; serological detection is mainly based on immunofluorescence, enzyme-linked immunoassay and other methods. Although these methods are fast, they lack specificity. It is easy to cause false positives; molecular biology detection mainly uses polymerase chain reaction (polymerase chain reaction, PCR) technology, which has the advantages of sensitivity, specificity, automation, etc., but there are also problems such as single detection is prone to false positives

Method used

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  • Borrelia burgdorferi detection kit and application thereof
  • Borrelia burgdorferi detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Design and synthesis of primers

[0024] The design of specific detection primers, after determining the target genes, download the sequences of all target genes from the genebank, use biological software for sequence comparison, intercept the most consistent sequence design primers, and perform blast comparison on the designed primers on the genebank, Verify the conservation of the primers. Primers were provided by Sangon Bioengineering (Shanghai) Co., Ltd.

[0025] Several pairs of primers were designed, and after experimentation, the final detection primers were determined as follows:

[0026] Borrelia Lyme disease OspC F: 5´- TAA TGA AAA AGA ATA CAT TAA GTG-3´

[0027] R: 5´-TTA AGG TTT TTT TGG ACT TTC TGC-3´.

Embodiment 2

[0028] The establishment of embodiment 2 bacterial strain template library

[0029] Bacterial strains were enriched and cultured according to the traditional culture method; 1 mL of the bacterial culture solution of the bacterial strains was taken, and the bacterial DNA was extracted with a bacterial genome DNA extraction reagent (Promega), and stored at -20°C for later use for detection; the established reference strain DNA template library, For specificity, sensitivity and precision tests.

Embodiment 3

[0030] Example 3 PCR general amplification

[0031] Use the exact same template to react with the same conditions for each primer pair, and select the primer with the best reaction effect; then optimize the Taq enzyme, primer dosage and cycle conditions until the following optimal PCR general reaction system and reaction conditions are obtained , the reaction system is as follows:

[0032] 10×Buffer (Mg 2+ plus) 2.5µl

[0033] dNTP (2.5mM each) 2µl

[0034] DNA 5µl

[0035] Primer (20pmol / µl) 1µl

[0036] TaKaRa Ex Taq (5U / µl) 0.25µl

[0037] wxya 2 O make up 25 µl

[0038] PCR reaction conditions: pre-denaturation at 95°C for 3min, denaturation at 95°C for 1min, annealing at 52°C for 50s, extension at 72°C for 1min, 30 cycles, and extension at 72°C for 10min; the reaction was completed at 4°C for the reaction product, and 5µL of the PCR product was washed with 1% agarose Gel electrophoresis detection.

[0039] According to the factors affecting the PCR reaction cond...

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Abstract

The invention discloses a Borrelia burgdorferi detection kit. The Borrelia burgdorferi detection kit comprises a PCR (polymerase chain reaction) primer for detecting Borrelia burgdorferi, and a Borrelia burgdorferi detection system is provided; a bacteria solution is diluted 1/10<8> times in a PCR-immune colloidal gold test strip detection method, a detection band changes color obviously, a positive result can be judged clearly, and the detection kit is proved to be higher in detection sensitivity; by combining a high-sensitivity and high-specificity method of PCR in nucleic acid testing withan immune colloidal gold rapid testing technique in immunological detection and designing the unique primer, extracted target DNA is subjected to specific amplification, an amplification product in adeveloping solution binds with a gold labelled antibody fixed on the test strip, a stable and visible detection band and a quality control band are formed, and rapid detection of Borrelia burgdorferiis realized.

Description

technical field [0001] The invention belongs to the fields of molecular biology and immunology, and in particular relates to a Borrelia burgdorferi detection kit and application thereof. Background technique [0002] Lyme disease (Lyme Disease, LD) is a natural foci disease caused by Borrelia burgdorferi discovered in the 1970s and mainly transmitted by hard ticks. Lyme disease can cause multi-system and multi-organ damage, clinical manifestations are migratory erythema, neuritis, myocarditis, etc., and severe cases can cause disability or even death. At present, Lyme disease has been reported in more than 70 countries in the world, and the incidence rate is on the rise. In 1992, the World Health Organization listed this disease as a key object of prevention and treatment, and this disease has become a global public health problem. In recent years, more than 10,000 cases have been reported every year in China, and its harmfulness has attracted people's attention day by day...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804C12Q1/04
CPCC12Q1/6804C12Q2531/113C12Q2565/625Y02A50/30
Inventor 王玮琳聂丹丹孙舒刘阳刘韬杨帆杨璐罗雁非刘金华
Owner 吉林出入境检验检疫局检验检疫技术中心
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