Study On The Detection Of PCR-Amplified DNA Of Legionellae By Enzyme-Linked Immunoassay

Objective Serve actue respiratory syndrome (shorted for SARS) is a huge disaster in our country in 2003. Its clinical symptom is highly similar with legionellae pneumonia. It is very valuable and economic to diagnose legionellae pneumonia early and exactly. The suspicious case of SARS can be dropped to the lowest level. At present, a legionellae's culture can be diagnosed definitely. But it is difficult to culture legionellae and the sensitivity is poor. Alternative approach such as the detection of antigens (DFA), antibodies of legionellae also have poor sensitivity and specificity. Recently, the technology based on molecular, especially PCR technology, shows high sensitive and specific in the detection of legionellae. But the results estimate of the followed analysis method of PCR products, such as gelose and SDS-PAGE electrophoresis, is too subjective. And the method could be affected by a lot of factors, it exits toxic compound in the procedures, ultraviolet is harmful to bodies; moreover large swatch is difficult to deal with at the same time, etc. The method of Dot blot use nucleic acid probe, but the results estimate is subjective, too. The method of Isotope probe and fluorescence probe have high sensitivity and specificity, but isotope probe has radioactivity, the whole process costs a long time. Fluorescence probe need fluorometry, the reagent of fluorescence is expensive. So we establish a sentive, specific, rapid and simple method to detect legionellae by a liquid phase hybridizations for PCR-ELISA. Methods Principle of liquid phase hybridizations: legionellae gene of the 16S rRNA was amplified by bio-labeled up-stream primers and general down-stream primers. One strand of DNA of PCR products was labled with biotin. The products of PCR were denatured, and added into the streptavidin-coated wells, then the biotinylated products were captured. The specific hybrid DNA with biotin and digoxigenin was formed. Then the horseradish peroxidase antidigoxigenin conjugate was linked with the digoxigenin of the hybrid DNA. Finally the substrate was added and the OD were measured. 1. Two 20-base oligonucleotides were used as primers enclosing a 386-bp fragment of the 16S rRNA gene. L451: 5′-AGGGTTGATAGGTTAAGAGC-3′was located at positions 451 to 470, and L817: 5′-CCAACAGCTAGTTGACATCG-3′was complementary to positions 836 to 817. PrimerL451 was 5′biotinylated. We amplified Lp1-8, Lp12, Lp14, Ld, Lb, Ll. After PCR, 10μl amplification product was detected by gel electrophoresis. 2. The 5′-digoxigenin-labeled 20-mer probe 5′-CAACCAGTATTATCTGACCG-3′, hybridize with PCR product of Lp1-8, Lp12, Lp14, Ld, Lb, Ll. 3. Liquid phase hybridizations for PCR-ELISA: Microplate wells coated with 5μg/ml streptavidin, the double-stranded PCR product was denatured at 100℃, 5min, immediately at ice water, 5min, 10μl of...