Design For The Chip Labelled With Multi-molecular Beacon Probe And The Exploration On Multi-sites Detection Of Bacillus Tuberculosis

Objective:The current detecting methods for Bacillus Tuberculosis (TB) include many techniques such as X-ray, sputum smear, cell culture, medicine sensitivity, PCR, and so on. They are tedious or time-consuming methods with low sensitivity and specificity, which may easy lead to misdiagnosis and economic overburden of patients. A great variety of drugs prescribed to patients with Tuberculosis and their overuse lead to the increasingly complicated mutation of drug-resistant genes with wide complex range of mutation sites from the single-base mutation, deletion, insertion, malposition to that with multiple-sites and large fragment which present a barrier to detecting the drug resistant mutation genes accurately and comprehensively. Especially for the test for wide-existing single- base mutation sites, the traditional techniques including the traditional gene chip can hardly address the problems. The multi-drug-resistance is the main factor affecting its treatment and popularity for its simple indicator and scarce information on drug-resistance. Thus to construct a system for identifying the TB and testing it drug resistance in a fast, sensitive, comprehensive and simple way is a issue urgent to addressed in the field of Laboratory Diagnostics both at home and abroad. The molecular beacon probe is a nucleic acid probe with great capacity of testing single-base mutation sites, however, due to its molecular limitation, there still is blank in its practice application in the fluorescence chips.In this study, we attempt to construct a chip technique labelled by MB of TB multiple resistance genes mutation by making advantage of the stronger specificity to detection of the single–base mutation sites of target molecular and the beacon probe, combining with the high throughput of fluorescence, which is capable of directly detecting the specificity of multiple target sites of drug resistant genes and the nucleic acid of TB and provides a new method to identify the drug resistant genes of TB and to make the epidemic investigation.Methods and Materials:1. Based on the IS986 gene sequence of TB found by GenBank, we designed the primers on the software Primer Premier V5.0. After optimizing the MgCl2, primer, dNTR concentration and the annealing temperature, we constructed the TB PCR reaction amplification system and made a comprehensive assessment on the system by sequencing and detecting its sensitivity and specificity. The generally used molecular beacon probe of TB was designed on the software of beacon designer 2.1. After the temperature, time and means in hybridization were optimized, we added into TB beacon probe for hybridization during and after PCR reaction. The fluorescent signal of MB-PCR products and its observing condition were explored by using various instruments such as the Image Acquisition and Analysis System, Fluorospectrophotometer, and Fluorescence Microscope. We made careful observation on the various fluorescence quenching molecular.2. By studying the connection of molecular beacons with one-side extending arm and the connection between the biotin and avidin, we explored on the fixation and hybridization means of TB molecular beacon and the chips. The optimizing experiments on enhancing the hybrid efficiency of molecular beacon chips include: the concentration of 5/oligonucleotide probes and molecular beacon probes, the temperature, duration time and the formula of hybridization, the spotting concentration and the size, the critical value distinguishing the positive and negative. The clinical application of TB-MB chips that were generally used and methodological comparison were also made.3. The main mutation regions of drug resistance in 4 kinds of TB were searched for in the NCBI database. Based on the above sites, we designed the proliferation system and optimized it. The molecular beacon probes and the probes with one-side extending arm were also designed on the basis of the sites. The following series of experiments on the surface of chips molecular beacon probes with one-side extending arm of the mutation sites of TB resistance: the hybridization temperature gradient investigation (45℃,50℃,55℃,60℃,65℃were selected), the hybridization duration gradient investigation( the duration time was made as 2,4,6,8,10,12,14,16,18h), the target sequence hybridization concentration gradient investigation ( the concentration selected were 0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09uM). The observation was made by using Fluorescence Microscope.4. The PCR proliferation of the responsive clinically isolated mycobacterium tuberculosis was made on the basis of above sites. The sequencing of the products was made. The PCR products at each site were hybridized by using the multiple MB chip under the optimized condition we explored and the results of sequencing were made comparison.Results:1. The MgCl2, primers, and the best concentration of dNTP in the generally used TB-PCR reaction system after optimization were: 4mmol/l,0.04μmol/l and 0.2 mmol/l respectively, the annealing temperature was 55℃. The result of sequencing fully agreed with that published by GenBank. No cross-reaction with other bacteria in the system. The testing limitation was 1 copy. The optimized hybridization temperature and duration time for water bath, the PCR instrument, the constant-temperature shaking table were 55℃,16h;65℃,4h;50℃,7h respectively. The significant difference of fluorescence intensity between the positive MB-PCR hybridization products and negative ones was observed by using Fluorescence Microscope(p<0.01). The high efficiency of the fluorescence quenching was showed in Cy3-BDH,FAM-DABCLYE, and low efficiency in Cy3-DABCLYE,FAM–BDH.2. It is difficult to modify the stem arm biotin-avidin of molecular beacon. The marking is made in middle of quenching molecular in the molecular beacon with one-side extending arm and the single extending arm with stem structure is capable of fixing well on the chips with high specificity and low difficulty in modifying. The best concentration of oligo probes is 40μmol/L. After comparing the hybridization results of the three fluids, we found that 0.15%SDS showed better result than 0.2%SDS and 10×SSC showed better result than 5×SSC. While the concentration of Cy3-BDH probe was 15μmol/L and that of FAM-DABCLYE probe was about 9μmol/L, the strongest signal was found after 7h hybridization reaction. As the spotting diameter diminished, the fluorescence intensity for the sample with same concentration remained the unchanged. While the concentration reduced, the intensity diminished. The ideal intensities distinguish the positive and negative signals are about 10μmol/L for Cy3-BDH, about 15μmol/L for FAM-DABCLYE. The total detection rate for clinical samples by using the TB-MB chips was 57.5%(23/40), which is 27.5%(11/40) higher than the smear analysis and 35%(14/40) than the method of culture. The statistically significant difference was showed by comparing the three ways of dictation. (χ2=7.366,P<0.01;χ2=32.281 , P<0.01 respectively).3. The main mutation regions of drug resistance in 4 kinds of TB were accurately identified. Resistant streptomycin(SM):rpsl(43,88), rrs;Resistant isoniazid (INH):katG(315,463);Resistant Ethambuto (lEMB):embB(285,303,306,330);Resistant Ethambuto (lEMB):the core area of ropB mutation; the extended mutation area. Based on the above sites, we designed the proliferation system and optimized it. The fragments with specificity were proliferated successfully. The molecular beacon probes and the probes with one-side extending arm were also designed on the basis of the sites. A series of hybridization experiments on the surface of chips molecular beacon probes were successfully conducted. In the order of rpsl43,rpsl88,rrs,katG315,katG463,embB285,embB303,embB306,embB 330,ropB-MB1-1,ropB-MB1-2,ropB-MB1-3,ropB-MB1-4,ropB-MB2-1,ropB-MB2-2, the best hybridization conditions were: 50℃,50℃,50℃,55℃,65℃,55℃,55℃,45℃,60℃,60℃,50℃,65℃,60℃,55℃,55℃(in the hybridization temperature gradient investigation), 12,14,14,16,12,12,8,14,10,10,10,8,12,10,8h(the duration time for the hybridization duration gradient investigation), 0.06,0.05,0.06,0.04,0.05,0.04,0.07,0.05,0.05,0.05,0.06,0.04,0.05,0.04,0.04uM (in the target sequence hybridization concentration gradient investigation). A statistically significant difference between the fluorescence positive signals and negative ones at above sites was observed.4. The PCR proliferation of the responsive clinically isolated mycobacterium tuberculosis was made successively on the basis of above sites. The results for the proliferation products sequencing were: the mutation rate of the resistant streptomycin(SM)rpsL genes codon 43 was 65%, codon 88 was 60% with 75% mutant strains overlapping with codon 43; the mutation rate of Rrs was 15% and the multiple sites repetition rate of resistant SM was considerably high; the mutation rate of the resistant Resistant isoniazid (INH) KatG315codon was 50% and that 463 was 37%; the mutation rate of the Resistant Ethambuto (lEMB) genes codon 285 was 15% and those of 303,306, and 330 were 6%, 70% and 3% respectively, and that of 306 was highest. All the mutation sites of resistant rifampicin rpoB were found and showed completed and more simultaneous mutation in multiple sites. The PCR products at each site were hybridized successfully by using the multiple MB chip under the optimized condition we explored. The results of good consistency in sequencing comparison were showed.Conclusion 1.In this study, we successfully made detection on the multiple targets of the main mutation genes with drug resistance of 4 TB by using the multiple molecular beacon probes. The design model and its hybridization techniques of the molecular beacon probes with high specificity and sensitivity to the single base targets were explored.2. We designed the MBs with high specificity generally used in detection of TB and used them in a series of experiment of proliferation and after proliferation. We also used the various instruments such as the Image Acquisition and Analysis System, Fluorosp ectrophotometer, and Fluorescence Microscope to explore the fluorescent observation condition of hybridization by using MBs, which reserved a technique for application of fluorescent chips. By comparing with the traditional ways to detest the clinical sample of TB, we proved that the TB-MB fluorescent chips have high detection rate, specificity and sensitivity.3. The design of molecular beacon probes with one-side extending arm has provided a simple idea to solve the problem of fixation of probes on the surface of chips, which is of significance of application of traditional MBs into the high density chips which can not be achieved for its difficulty in stem arm modification and the absence of specificity.4.The PCR proliferation system of the mutation genes on the basis of sites of 4 types of TB was successfully made. The responsive multiple TB molecular probes and its probes with one-side extending arm were synthesized, designed and screened. A series of experiments on the hybridization of MB probe chips and its optimization was also made.5. The PCR products at each site were hybridized successfully by using the multiple MB chip under the optimized condition we explored. The results of good consistency in sequencing comparison were showed. It indicated that the molecular beacon probes have high specificity and sensitivity to the single base mutation. If used in the genes chip fields, the combined detection for the multiple targeted single base mutation could be achieved.