Study On Gene Diagnosis Of Commonly Encountered Pathogenic Bacteria In Lower Respiratory Tract Infections

Objective: To establish a dot-blot hybridization technique for rapid detection of commonly encountered pathogenic bacteria in lower respiratory tract infections through the long specific DNA probes of pathogenic bacteria detecting bacterial DNA labeled with biotin directly. The technique is established according to the principle of DNA bases match and the feature of different-origin complementary single-stranded DNA forming double-stranded DNA under the suitable circumstances. The method is used to detect clinical sputum specimens and the results are compared with those of sputum culture and polymerase chain reaction to evaluate the diagnostic value of it in lower respiratory tract infentions. Methods: 1. Streptococcus pneumoniae, haemophilus influenzae, methicillin-sensitive staphylococcus aureus, methicillin-resistant staphylococcus aureus, methicillin-sensitive staphylococcus epidermidis, methicillin-resitant staphylococcus epidermidis, pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, legionella pneumophila were chosen as identified bacteria. The long specific DNA probes were synthesized by polymerase chain reaction in the highly specific gene sequences of above 10 bacteria. The species-specific probes synthesized were inoculated on a nitrocellulose filter and hybridized with template DNA labeled with long-arm photosensitive biotin. The signal of hybridization reaction was observed to detect which bacteria infected. The sensitivity and specificity of the method were studied. 2. The dot-blot hybridization was used to detect sputum samples from 100 community-acquired pneumonia patients, 150 hospital-acquired pneumonia patients and 50 non-lower respiratory tract infections patients. The results of hybridization were compared with those of sputum culture and polymerase chain reaction. Results: 1. The eleven DNA probes synthesized were highly specific. Each specific probe only hybridized with its corresponding bacterial DNA without cross reaction with other bacteria, fungi or viruses. 2. The sensitivity of the probes was 0.2ng. 3. The dot-blot hybridization was capable of detecting Ing-5-bacterial DNA. 4. The dot-blot hybridization could detect mixed infections of more than one causal agent. 5. The positive rates of dot-blot hybridization and culture in detecting 100 sputum samples of community-acquired pneumonia patients were 53% and 29% respectively. The difference was statistically significant (P < 0.01). The positive rate of PCR was 90% and was higher than that of hybridization (P < 0.01). 6. The positive rates of dot-blot hybridization, culture and PCR in detecting 150 sputum samples of hospital-acquired pneumonia patients were 62.7 % , 38 % and 93.3 % respectively. The positive rate of hybridization was higher than that of culture (P < 0.01) while the positive rate of PCR was higher than that of hybridiztion (P < 0.01). 7. The positive rates of dot-blot hybridization, culture and PCR in detecting 50 sputum samples of non-lower respiratory tract infections patients were 6%, 8% and 60% respectively. The difference between sputum culture and hybridization was not statistically significant (P > 0.05). The positive rate of PCR was higher than that of hybridiztion (P < 0.01). Conclusions: 1. Detection of pathogenic bacteria in lower respiratory tract infections through the long specific DNA probes of pathogens hybridizing with template DNA is an applicable method. 2. The positive rate of hybridization is higher than that of sputum culture in the etiological diagnosis of community-acquired pneumonia. Hybridization is especially fit for detection of bacteria that are diffult to culture or need a long time to culture. 3. The positive rate of hybridization is higher than that of sputum culture in the etilolgical diagnosis of hospital-acquired pneumonia, especially for those patients who have received antibiotics before sputum samples are taken. 4. The dot-blot hybridization is proved to be a sensitive and specific method that can be used for rapid diagnosis of lower respiratory tract infections. I...