Application of Representational Difference Analysis in Cloning Mycoplasma Arthritis Specific DNA Fragment

• Main Author: 郭瓊穗
• Other Authors: 邱卓凡
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/10092572170805607614

碩士 === 國立臺灣大學 === 微生物研究所 === 84 === Mycoplasmas are the pleomorphic, cell well-less and the smallest bacteria. They are found in the animal tissues, including pleura, peritoneum, respiratory tract, synovia joint, urethra, genital tract, and middle ear. Mycoplasma arthritidis and Mycoplasma pulmonis are two pathogens of rat and mouse. Both of them can cause clinical syrndoms of animals including bronchitis, otitis media, laryngitis, tracheitis, alveolitis, conjunctivitis, chronic polyarthritis, murine respiratory mycoplasmosis (MRS) and urethritis, and sometimes, the pulmonary abscess and pleuritis. The mycoplasma infected animals will result in the reduction in productivity and cause the economic loss. It also has a scientific impact if a research was done by using mycoplasma infected rat or mouse as experimental model. Therefore, a good quality of experimental animals used in researches is the highest priority requirement. However, M. arthritidis and M. pulmonis can cause the similar clinical syrndoms and pathological lesions. They have antigenic cross reaction in serological diagnosis, and the homology in their DNA sequences is also high. These characters make a difficulty for us to differentiate between these two organisms.The purpose of this study is to clone a M. arthritidis specific DNA fragment by representational difference analysis (RDA) which was reported by Lisitsyn in Science volume 259: 946-951, 1993. In this study, a DNA fragment containing 194 bp, named MA194 was obtained. This DNA fragment was proved to be M. arthritidis specific by DNA hybridization with all testing organisms genomic DNA. Only by using M. arthritidis genomic DNA was shown to be a positive result. The DNA sequence of this fragment was as following: 5'-AAGCTTGCATTGCTAGAGG TGGATGTAGTTGTCGGTTCTTTTGCAACTAATTTTTCAATTT CTTCTAGTAGTTTTTTAGCATCTTCTTTAAGTTTATCTTCTCT ATCTTGAAGCATTTTTAGTTTATCTTTAGCTTCTTTTAAGGTT TCTTTAGACAGATCAATAGTTGTAGCAAGCTCTTCTGCTAA AAGCTT-3'. We used the DNA sequence to design a pair of primers sequence for polymerase chain reaction (PCR) to verify the specificity of MA194. Primer sequences were as following: 5'TAGAGGTGGA TGTAGTTGTCGGTT-3' and 5'-GCAGAAGAGCTTGCTACAA CTATT-3'. None of oganisms except M. arthritidis genomic DNA as template had a PCR product. For investigation of mycoplasmas infection in rats of animal center. National Taiwan University, College of Medicine, PCR and cultivation methods were used in the study. From the 121 rat tracheal segmented specimens, a 38.0 % positive rate was obtained by cultivation for mycoplasmas without species specification and a 48.8% positive rate was obtained by PCR detection for M. arthritidis alone.