Detection of oprI gene of Pseudomonas aeruginosa by reverse dot-blot hybridization

Abstract

Abstract: Objective To establish a rapid method for detecting Pseudomonas aeruginosa at the early stage of infection. Methods Specific primers were designed according to oprI gene sequence of Pseudomonas aeruginosa, and the specific probe was synthesized by PCR. After photosensitive biotin labeling of the bacterial DNA, reverse dot-blot hybridization was used to detect Pseudomonas aeruginosa. Results The probe synthesized was highly specific to Pseudomonas aeruginosa without cross reaction with other bateria, viruses or fungi. The method was capable of detecting 100 ng bacteria DNA. Conclusion Reverse dot-blot hybridization possesses the merits of speediness and specificity in the detection of Pseudomonas aeruginosa in the early stage of infection.

CLC Number:

FAN Hui-zhen¹, HUANG Wen-jie¹, LIANG Kun¹, FANG Yi¹, MA Li-ren², LIU Yao-qing². Detection of oprI gene of Pseudomonas aeruginosa by reverse dot-blot hybridization[J]. Journal of Southern Medical University, 2004, 24(03): 303-305.

References

[1] Saint Onge A, Romeyer F, Lebel P, et al. Specificity of the Pseu domonas aeruginosa PAO1 lipoprotein I gene as a DNA probe and PCR target region within the Pseudomonadaceae [J]. J Gen Microbiol, 1992, 138(Pt4): 733-41.
[2] 奥斯伯,布伦特,金斯顿,等著.颜子颖,王海林译.精编分子生物学实验指南[M].北京:科学出版社.1998.39.
[3] 黄媛,牟兆钦,陈建魁,等.PCR结合寡核苷酸探针杂交检测临床常见真菌的实验研究[J].中华微生物学和免疫学杂志.2001,21(3):345-8.Huang Y, Mu ZQ, Chen JK, et al. Detection and identification of fungi with clinical significance by PCR combined with oligonucleotide probe hybridization [J]. Chin J Microbiol Immunol, 2001,21(3): 345-8.
[4] 尚世强,洪文澜,俞惠民,等.细菌DNA的聚合酶链反应扩增及反相杂交初步分型[J].中华检验医学杂志,1998,21(5):281-4.Shang SQ, Hong WL, Yu HM, et al. Amplification of bacterialDNA by polymerase chain reaction(PCR) and typing by reverse hybridization[J]. Chin J Clin Lab Med, 1998, 21(5): 281-4.
[5] 王卫华,肖红,肖勇,等.长臂光敏基因探针对结核性浆膜腔积液的诊断价值.中华检验医学杂志[J].2001,24(2):79-81.Wang WH, Xiao H, Xiao Y, et al. The diagnostic value of improved PCR in pleural offusion tuberculosis[J]. Chin J Clin Lab Med, 2001, 24(2): 79-81.
[6] 于润江.高龄者难治性细菌性呼吸道感染的对策[J].中华结核和呼吸杂志,2001,24(6):335-8.Yu RJ. Measure of intractable bacterial respiratory infection in oldman [J]. Chin J Tuberc Respir Dis, 2001, 24(6): 335-8.
[7] 陈民钧,王辉.中国重症监护病房革兰阴性菌耐药性连续7年监测研究[J].中华医学杂志,2003,83(5):375-80. Chen MJ, Wang H. Continuous surveillance of antimicrobial resis tance among nosocomial gram-negative bacilli from intensive care units in China[J]. Natl Med J Chin, 2003, 83(5): 375-80.
[8] Osmon S, Ward S, Fraser V J, et al. Hospital Mortality for Patients With Bacteremia Due to Staphylococcus aureus or Pseudomonas aeruginosa[J]. Chest, 2004, 125(2): 607-16.
[9] Hancock RE, Siehnel R, Martin N. Outer membrane proteins ofpseu domonas [J]. Mol Microbiol, 1990, 4(7): 1069-75.
[10] Larbig M, Mansouri E, Freihorst J, et al. Safety and immunogenicity of an intranasal Pseudomonas aeruginosa hybrid outer membraneprotein F-I vaccine in human volunteers [J]. Vaccine, 2001, 19(17- 19): 2291-7.
[11] Vaughn CP, Elenitoba-Johnson KS. Hybridization-induced dequench ing of fluorescein-labeled oligonucleotides: a novel strategy for PCR detection and genotyping[J]. Am J Pathol, 2003, 163(1): 29-35.
[12] 李凌,马文丽,毛向明,等.HIV基因芯片的初步研究[J].第一军医大学学报,2002,22(8):724-7.Li L, Ma WL, Mao XM, et al. Preliminary study of development of gene chips for HIV diagnosis[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(8): 724-7.
[13] 张灵霞,庄玉辉,杨华卫,等.两种DNA探针杂交检测结核分枝杆菌的研究[J].中华检验医学杂志,2001,24(2):106.
[14] 吕梁,马文丽,王洪敏,等.应用PCR快速制备细小病毒B19诊断芯片探针[J].第一军医大学学报,2003,23(11):1121-4.Lv L, Ma WL, Wang HM, et al. Quick preparations of human parvovirus B19 microarray probes using PCR [J]. J First Mil Med U niv/Di Yi Jun Yi Da Xue Xue Bao, 2003, 23(11): 1 121-4.
[15] 张宝,马文丽,石嵘,等探针的纯化与否对基因芯片重复利用的影响[J].第一军医大学学报,2002,22(3):208-10.Zhang B, Ma WL, Shi R, et al. Effect of probe purification on the reutilization of gene chip [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(3): 208-10.