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Rapid detecting room dust mite allergen second group constituent content florescent real time ration PCR method

A component content, real-time quantitative technology, applied in the field of allergen detection, can solve the problems of inability to separately detect the concentration of dust mite allergen components, slow detection speed, high cost, etc., to achieve the prevention of dust mite allergic diseases, strong and practical Value, accurate results

Inactive Publication Date: 2017-02-22
YANCHENG INST OF HEALTH SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The RAST inhibition test measures the content of dust mite allergens in the air, but the RAST inhibition test is the detection of the total activity of dust mite allergens, and it cannot detect the concentration of various allergen components in dust mite separately, and its cost is relatively expensive and expensive. There is environmental pollution
The radioimmunoassay (RIA) method was used to detect the common antigen (antigen P1) of group I allergens of Dermatophagoides farina (Der f) and Dermatophagoides pteronyssinus (Der p) to realize the typing of dust mites and overcome the The RAST method cannot detect the defects of the allergen content of different groups of dust mites, but it also has the disadvantages of radioactive isotopes having half-life and polluting the environment.
The double-antibody sandwich method of ELISA is widely used, but its cost is expensive and the detection speed is slow. It is mostly used in scientific research and is not suitable for the widespread detection of dust mite allergen content in house dust in epidemiological investigations.

Method used

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  • Rapid detecting room dust mite allergen second group constituent content florescent real time ration PCR method
  • Rapid detecting room dust mite allergen second group constituent content florescent real time ration PCR method
  • Rapid detecting room dust mite allergen second group constituent content florescent real time ration PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1 Preparation of Der f 2 Plasmid Standard

[0013] Design primers based on the Der f 2 nucleic acid sequence published by the International Gene Bank (GenBank AB195580), and add BamHI / Xho I restriction sites, synthesized by Dalian Bao Bioengineering Co., Ltd. as follows: Upstream primer F: 5′-GGATCCATGATTTCCAAAATCTTGTGCC -3′ (SEQ ID No: 1); Downstream primer R: 5'-CTCGAGTTAATCACGGATTTTACCATGG-3' (SEQ ID No: 2). Use High Fidelity PrimeScript™ RT-PCR kit (Code No. DR027A) for reverse transcription, and PrimeSTAR HS DNA Polymerase (Code No. DR010A) for PCR amplification. The full amount of the product obtained by the above RT-PCR was loaded into electrophoresis, the PCR product was recovered by cutting the gel, added with poly-A tail, refined, ligated with the pMD19-T plasmid vector, and thermally transformed into E.coli competent cell JM109. Spread the plate and incubate overnight at 37°C. After selecting positive colonies for enlarging and culturing, the plasmid ...

Embodiment 2

[0014] Example 2 Real-time fluorescent quantitative PCR primers and TaqMan probe design of Der f 2

[0015] Using Primer Express 2.0 software and following the TaqMan probe design principles, Der f 2 fluorescent quantitative PCR primers and TaqMan probes were designed according to GenBank (AB195580) sequence. See Table 1.

[0016] Table 1 Der f 2 primer and probe sequence.

[0017]

Embodiment 3

[0018] Example 3 Der f 2's real-time fluorescence quantitative PCR standard curve

[0019] The plasmid samples identified as Der f 2 recombinant positive plasmids by sequencing were measured on the DNA / RNA quantifier to determine the plasmid concentration of the samples, and the plasmid copy number was calculated according to the calculation formula reported in the literature. Plasmid copy number (copies / μL) = (recombinant plasmid concentration ng / μL×10 -9 ) ×6.02×10 23 / (660 × base number of recombinant plasmid).

[0020] Real-time fluorescent quantitative PCR detection program: According to a certain sequence, add the following components to the 0.1ml PCR tube: 10μL 2X Realtime PCR Master Mix, 1μL diluted Der f 2 plasmid standard, 2μL primer probe MIX(F / R / P each 10μM), 7μL 0.1% DEPC water, 20μL total system. Use the following procedures to implement the real-time fluorescent quantitative PCR program, 95 ℃ pre-denaturation for 1 min; 95 ℃ denaturation for 15 s; 60 ℃ annealing a...

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Abstract

The invention discloses a rapid detecting room dust mite allergen second group constituent content florescent real time ration PCR method. The method utilizes Der f 2 reorganized granule as the standard, designing the Der f 2 real time florescent ration PCR primer, and TaqMan probe, adopting the real time florescent TaqMan probe ration PCR method, through the construction of Der f 2 real time florescent ration PCR standard curve, to realize a rapid ration detection of the room dust mite allergen Der f 2 content. The method applying the environmentally friendly and cheap Der f 2 real time florescent ratio PCR method and commercialized expensive ELISA reagent kit respectively conducts detections on the same room dust samples, the correlation coefficient between the two methods is R = 0.81, P< 0.05, implying the linear relationship of the two methods is excellent.

Description

Technical field [0001] The invention relates to the technical field of allergen detection, in particular to a fluorescent real-time quantitative PCR method for rapidly detecting the content of the second component of dust mite allergen in room dust. Background technique [0002] At present, the detection of dust mite allergen content in indoor environment in my country is not very popular. Clinically, it is mainly used to diagnose patients through in vivo methods such as skin tests or in vitro methods such as detection of specific IgE antibodies in patients' serum, or use infrared rays The scanning detection system detects allergens related to the patient's medical history. But these methods can only play a diagnostic role, not a preventive role. Since the 1980s, scholars at home and abroad have successively established radioallergosorbent test (Radioallergosorbent Test, RAST) inhibition test, radioimmunoassay (RIA), and ELISA double antibody sandwich method for quantitative det...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2561/101C12Q2545/114
Inventor 俞黎黎崔玉宝滕飞翔张承伯杨李
Owner YANCHENG INST OF HEALTH SCI
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