Investigation On The Design Of High Specific Molecular Beacon Probe And Applying The Fluorescence Chip On Bacillus Tuberculosis

Objective:The current detecting method for Bacillus Tuberculosis (TB) include many techniques such as sputum smear, cell culture, PCR, and so on. It was a tedious and time-consuming method with low sensitivity and specificity, which may lead to misdiagnosis. Fast, convenient and sensitive TB detecting methods were required for the therapy and control of related diseases. In this investigation, we combined the techniques of biochip with molecular beacon probe, which was highly sensitive and specific, to establish a fluorescence chip of molecular beacon probe for high throughput detection of TB. Conditions for the fixing of molecular beacon probe to the array and detecting of fluorescence were optimized.Methods:1. DNA IS986 (Gen Bank) was selected as the target molecule. The primer was designed with Primer Premier V5.0. Parameters, specificity and sensitivity of TB-PCR system were optimized.2. Molecular beacon probe for TB PCR product was designed with Beacon designer2.1. A method for hybridizing probe with target molecule in PCR system was developed, and conditions such as concentration of molecular beacon probe, MgCl2, primer, dNTP, hybridizing temperature, time and style were optimized.3. The fluorescent signal of hybridization product was detected by fluorescent spectrophotometer and fluorescent microscope. Fluorescent molecule and quench molecule were used for molecular beacon probe in the test.4. Traditional biotin-avidin connection mode and unilateral extended arm connection methods were compared for fixing TB molecular beacon probe to the chip.5. Hybridization efficiency of molecular beacon probe with unilateral extended arm on the chip was optimized by adjusting the concentration of 5/oligonucleotide, the concentration and purity of molecular beacon probe, hybridization temperature, hybridization time, the prescription of hybridization solution, the drop size of sample, the comparation of molecular beacon fluorescence signals of 4 molecules, and the threshold for positive and negative results.6. The clinical application and technology comparison of TB-MB chip were estimated.Results:1. A 245bp DNA band with the same sequence as IS986 was found on the agarose gel after the electrophoresis of PCR product of wild type TB, clinically isolated TB strain or clinical positive samples. The best concentration of MgCl2, primer and dNTP for PCR were 4mmol/l, 0.04μmol/L and 0.3mmol/l respectively, and the optimized temperature of renaturation was 55℃. No cross reaction with other bacteria was found, and the detection limit of TB-PCR was 10 copies of DNA/ul.2. The optimized concentration of molecular beacon , MgCl2, primer and dNTP in the TB PCR reaction system were 0.04μmol/L , 5mmol/l, 0.04μmol/L and 0.3mmol/l respectively.After PCR reaction, the optimized conditions of water-bath, PCR apparatus, homoisothermy rocking bed were 55℃for 6 h,55℃for 4 h, and 50℃for 7 h. Positive and negative results were: water-bath> homoisothermy rocking bed> PCR apparatus.3. The fluorescence intensity of positive and negative (loop-stem structure was closed) product of MB PCR hybridization and non-dissociative molecular beacon probe PCR product (control) was detected by fluorescent spectrophotometer with the ratio of 6.102/0.136/0.003. Obvious difference was observed under fluorescent microscope. The efficiency of fluorescence quenching was higher in samples of Cy3-BDH, and FAM-DABCLYE than in Cy3-DABCLYE and FAM–BDH.4. Unilateral extended arm molecular beacon using quenching molecular middle label and stem structure unilateral extended arm (one side was quenched) could be easily fixed onto the chip and easily modified specifically. It was a better method than Biotin-avidin based fixation since the later was non-specific and hard to be modified.5. The optimized hybridization parameters were as following: 40μmol/L 5/oligonucleotide, 0.15 %SDS, 5×SSC, 15μmol/L Cy3-BDH molecular beacon probe or 9μmol/L FAM-DABCLYE probe at 65℃for7h, 10μmol/L Cy3-BDH molecular beacon probe or 15μmol/L FAM-DABCLYE probe at 65℃for 17h. Sample spotting diameter had no effect on the fluorescence intensity while the sample concentration was related to the intensity.6. The identification efficiency of TB-MB chip was 57.5% (23/40), which was higher than sputum smear 27.5% (11/40) and culture 35% (14/40). Statistical analysis indicated that data of TB-MB chip was significantly different from those of sputum smear (χ2=7.366,P=0.006<0.01) and culture (χ2=32.281 , P=0.000<0.01). No false identification was found on TB-MB chip, while both sputum smear and culture identified 1 from 20 control cases.Conclusion:1. TB-MB PCR reaction system could identify TB quickly from TB type strain, TB clinical isolated strain and clinical sample. It was more specific and sensitive than normal PCR detection, which settled the base of the design of high specific molecular beacon probe fluorescence Chip on Bacillus Tuberculosis.2. The design and apply of unilateral extended arm molecular beacon probe initially solved the difficulty in molecular beacon technology applied on chip technology. The use of specific and sensitive molecular beacon on chip technology was artful.3. The technique on the detection of molecular beacon probe fluorescence Chip on Bacillus Tuberculosis was set up, including sample preparation, chip preparation, hybridization reaction, scanning and image analysis etc. To give consideration on various kinds of factor influencing unilateral extended arm molecular beacon probe, the conditions in the reaction system were optimized reasonably.4. TB-MB chip was specific and sensitive with high identification efficiency, which was confirmed by 40 clinical sputum samples with sputum smear and culture. Unilateral extended arm molecular beacon chip could be better applied on clinic detection.