Identification And Detection Of Dermatophagoides Farinae And Dermatophagoides Pteronyssinus By The PCR Technology

In recent decades, The prevalence of all sorts of allergic diseases such as allergic rhinitis, dermatitis, bronchus asthma and allergic conjunctivitis are increasing year by year[1], it has become a global health problem[2]. Allergic disease is an immune pathological reaction process produced by contacting allergens[3]. Existing literatures[4] prove that house dust mite is one of the most important inhalant allergens. And as a family of house dust mite, Dermatophagoides farinae (D.f) and Dermatogoides pteronyssinus(D.p) have the largest number, most widely distribution, they are considered as the most harmful to human health in dust mites [5-7]. But on one hand, the distribution of D.f and D.p are not completely the same, on the other hand, they often coexist in a certain environment, so understanding the kinds of dust mites and the dominant dust mites in a region or environment is very important to the diagnosis, prevention and treatment of allergic diseases. This study attempted to use Polymerase Chain Reaction (PCR) technology to identify and detect D.f and D.p.Objective:Because D.f and D.p are widely distributed in indoor environment, and they are very similar in morphology, sometimes, there are difficulties or troubles when they are identified or detected with traditional methods. therefore, this study attempted to use PCR technology to identify and detect them.Methods:(1) Preparation of D.f and D.p DNA The DNA of D.f and D.p were extracted with phenol-chloroform.(2) Randomly primed PCR (RP-PCR) amplification With the six randomly primers RP1, RP2, PR3, RP4, RP5 and PR6 alone or paired, both D.f and D.p DNA were amplified by PCR. Through analyzing the differences of amplification products between D.f and D.p we were going to understand if RP-PCR can be used in identifying the two dust mites.(3) Sequencing Three bands were selected from the RP-PCR amplification products, they were sequenced and analyzed in homology.(4) Specific primers design Designed specific PCR primers based on the sequencing results, used in the identification and detection of D.f and D.p.(5) PCR amplification of D.f and D.p DNA templates of D.f and D.p were amplified by PCR with the specific primers.(6) Specificity of PCR Using the specific primer of D.p amplified D.f DNA, and the D.f primer amplified D.p DNA, then observed the specificity of these primers.(7) Sensitivity of PCR Diluted the DNA template of D.f and D.p, then used the diluted DNA templates for PCR amplification to estimate preliminarily the sensitivity of this method.(8) Optimization of PCR condition By reducing cycle time and cycle number the two ways to optimize the PCR conditions and reduce the detection time.Results:(1) Preparation of D.f and D.p DNA The OD260 / OD280 of D.f and D.p DNA were 1.78 and 1.86 respectively, it showed that they have the purity to be further used in PCR.(2) RP-PCR amplification The results of the PCR at the nine different patterns (six primers alone or matching) showed that, the amplification products of D.f and D.p are different at some extent when used the same primer(s). It suggested that RP-PCR method can be used to identify the two dust mites. Among them, RP1,RP3 matching amplification, two bands around 500bp and 750bp were seen in amplified products of D.f; while only a clear band about 500bp in D.p and no product larger than 500bp appeared. So selected the PCR products for further research to identify and detect the two dust mites.(3) Sequencing Three DNA fragments size of 502bp, 666bp and 500bp were sequenced, and no highly homologous sequence discovered.(4) Specific primer design Three pairs of specific PCR primers were designed according to the sequencing result: two pairs of them f1 ,f2 and f3, f4 were D.f primers, whose product size expected were 478bp and 657bp respectively; while the product size of p1, p2 D.p primer will be about 328bp.(5) PCR amplification of D.f and D.p PCR amplification of D.f and D.p with f3, f4 and p1, p2 primers, the products as long as predicted were obtained, but no obvious bands appeared when using f1, f2 for PCR. So f3 and f4 and p1, p2 can be used to identify and detect the two dust mites.(6) Specificity of PCR The DNA of one dust mite were amplified by PCR with the specific primer of another dust mite, neither D.f nor D.p had significant bands. It showed these primers have good specificity.(7) Sensitivity of PCR With the specific primer, we can amplify the target bands from the DNA template about 1.5 dust mites by PCR, which suggested that this method has a good sensitivity.(8) Optimization of PCR condition Shorten the reaction time of the cycle, the target bands still appeared; when reduced the cycle number from 45 to 35 or 40, the amplification reaction was still successful.Conclusion:(1) The research results show that: by proper selection and match of random primers, RP-PCR technology established can be used in effective identification of D.f and D.p at gene level. (2) Three DNA fragments of D.f and D.p were sequenced and analyzed in homology, the results suggest these may be new gene fragments. In addition, two amplification products of D.f and D.p were the very close in size (about 500bp), but sequencing results revealed that they were not the same or similar DNA fragments.(3) The specific primers have better effects to PCR amplification of D.f and D.p DNA, and they have the potential in identification and detection of the two dust mites from the environmental samples.(4) By optimization of the condition, PCR detection time can be shortened further, and the aim of rapid detection was achieved.