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Identification and Detection of Dermatophagoides Farinae and Dermatophagoides Pteronyssinus by the PCR Technology

Author: DingXue
Tutor: ChenDaiXiong
School: Guangzhou Medical College
Course: Medical Immunology
Keywords: Farinae House dust mite PCR Distinguish Detect
CLC: R440
Type: Master's thesis
Year: 2011
Downloads: 29
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Abstract

Allergic diseases the contact allergen body to produce an immune pathological response process [3]. The largest number of dust mites Youyi dust mites (Dermatophagoides farinae, Df) and house dust mite (Dermatogoides pteronyssinus, Dp), the most widely distributed, most harmful [5-7]. But not exactly the same as the one hand, house dust mites and dust mite distribution, on the other hand, they often co-exist in the same environment, and therefore certain area or type of environment, dust mites and dominant mite species of allergic diseases the diagnosis, prevention and treatment is very important. This study attempts to polymerase chain reaction (Polymerase Chain Reaction, PCR) technology for dust mites and house dust mite identification and detection. Research purposes due to dust mites and house dust mites in the indoor distributed widely, and the two are very close in morphology to identify or detect by traditional methods difficult or more trouble, this study attempts to use the PCR method of dust mites and house for identification and detection of dust mites. Research methods: (1) dust mites and house dust mite DNA was extracted with phenol - chloroform method, respectively, to extract dust mites and house dust mite's of DNA (2) Random PCR amplification with the design of six random primers RP1, RP2, PR3, RP4 , RP5 and PR6 individually or pairwise matching, two dust mites DNA for PCR amplification. Than the same primers amplified two dust mites results verify that the random primer PCR method is able to identify two kinds of dust mites. (3) sequencing selected from the results of random primer PCR reaction DNA amplification bands sequencing and analysis of three identification and detection of dust mites and house dust mites potential. (4) PCR primers were designed based on sequencing results, specific primers were designed for the identification and detection of the dust mites and house dust mite. (5) PCR amplification of dust mites and house dust mites new design primers for PCR amplification of dust mites and house dust mites DNA. (6) PCR reaction specificity with a new design of the house dust mite primers the dust mites DNA, The dust mite primers house dust mites DNA observed specificity of these primers. (7) the sensitivity of the PCR reaction to dust mites and house dust mite DNA template dilution, take the diluted DNA template for PCR amplification reaction to preliminary estimates of the sensitivity of the detection method. (8) the optimization of the PCR detection method, by reducing the cycle in each portion of the reaction time and reduce the number of reaction cycles in two ways on the PCR reaction conditions were optimized to shorten the detection time required. Findings: (1) dust mites and house dust mite DNA extraction of dust mites and house dust mite get DNA OD260/OD280 respectively 1.86 and 1.78, indicating that it has a higher purity can be further used for PCR experiments. (2) Random PCR amplification of six primer alone or paired for PCR reaction amplification results than a total of nine cases of the visible, the same primers dust mites and house dust mite amplified bands there are different degrees of difference. Prompt random primer PCR method can be used for the identification of the two dust mites. Which, RP1, RP3 paired amplification, dust mites in the 500bp and 750bp were significantly bands about; house dust mite only 500bp bands clearer, no greater than 500bp amplified product. Therefore selected amplification product of this primer is further used for the identification and detection of two kinds of mites. (3) Sequencing of three sizes were measured respectively, 502bp, 666bp and 500bp DNA fragment sequence structure, homology analysis found no thereto highly homologous sequences. (4) PCR primers designed based on sequencing results, total design three pairs of specific primers material: two pairs amplified dust mites cited material f1, f2 and f3, f4, expected amplification product length 478bp and 657 bp, respectively, amplified by the house dust mite primers p1, p2 expected amplification product length of 328bp. (5) PCR amplification of dust mites and house dust mite f3 f4 p1, p2 primer PCR amplification product of the length and the expected size of dust mites and house dust mites and the f1, f2 amplified clear bands appear. Therefore, f3, f4, and p1, p2 can be used for the identification and detection of two kinds of dust mites. (6) PCR reaction specificity of the above-mentioned two pairs of primers cross PCR amplification, both dust mites or house dust mite no significant strip appears. Show that the primer has a good specificity. (7) PCR reaction with a sensitivity new design of primers for PCR can be amplified from the equivalent of 1.5 DNA template dust mites, Dermatophagoides farinae and Dermatophagoides pteronyssinus corresponding destination strip, indicating that the detection method has good sensitivity sex. (8) PCR detection method optimized to shorten the reaction time of the loop, dust mites and house dust mites can be amplified target bands; to reduce the number of cycles from 45 to 35 or 40, both can still be amplified the target band. Conclusion: (1) The results of this study show that: the selection of the appropriate random primers and the primer pair screening, random primer PCR method, from dust mites and house dust mites effectively identify the gene level. (2) Determination of the the three dust mites and house dust mite DNA fragments, by whom highly homologous sequence homology analysis without these three fragments may be a new gene fragments. In addition, dust mites and house dust mite by the same random primer amplification generated, size is very close (about 500bp) product sequencing showed that they are not the same or similar DNA fragments. (3) specific primers used for amplification of the two dust mites have good results, and has the potential of both the identification and detection from environmental samples. (4) PCR detection method optimization results suggest that the PCR detection of dust mites and house dust mite reaction time can be shortened by optimizing the rapid detection purposes.

Identification and Detection of Dermatophagoides Farinae and Dermatophagoides Pteronyssinus by the PCR Technology

Abstract

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