| BackgroundAlmost all of the studies about the Mycobacterium tuberculosis(MTB)were based on the discovery that the MTB was infectious as the pathogen of the tuberculosis(TB).After that finding made by Robert Koch in 1882,a variety of the MTB ralated methods for the prevention and treatment have been developed,however,the TB is still a severe globle public health problem so far.As per the report of World health organization(WHO),around 9 million individuals have underwent the infection of MTB each year all over the world(including incubation status and activation status,respectively).Further,about 1.5 million people have died from various types of the TB every year in the globle scope.Therefore,MTB is one of the primary lethal pathogens.The current phenomenons above have been due to the following points: a,the MTB has obtained a complicate mechanism of infection in the processing of evolution;b,the infection of human immunodeficiency virus(HIV)increases the risks of the combined infection of MTB;c,the numbers of new types of drug resistance and multiple drug resistance have been growing.It was reported that around 400 thousands individuals has been infected by the HIV and MTB simultaneously worldwide in 2014.Besides,about 480 thousands new cases of multiple drug resistance were detected out in 2013.In addition of the MTB,the Mycobacterium tuberculosis complex(MTBC)is also able to cause the TB,especially the pulmonary TB.Meanwhile,as the same as the MTB,the Mycobacterium avium complex(MAC)were isolated from the respiratory tract and related with the pulmonary infections.So it is critical to indentify the pathogens in the prevention and treatment for the TB regardless of the pulmonary infection or the extra-pulmonary infection.In general,the bacterial culture of the pathogen is the Gold Criteria for the diagnosis of the TB.Nevertheless,the pathogen culture method has not been uesd extensively due to the obvious disadva ntages such as the high time-cost and strict culture conditions requirement.Therefore,a series of biochemical tests and biomolecular investigations is needed to make certain which kind of the pathogens is the primary cause including the polymerase chain reaction(PCR),real time PCR(RT-PCR)and so on in general.Those so-called new methods have less time costs,but they possess higer technological levels,more analysis difficulties and risks of detection loss for the early imperceptible infection because of the amplification threshold.Hence,it will be of great importance in the prevention and treatment for the TB to establish a new test method which is more sensitive and rapid than the conventional approaches.Surface plasmon resonance(SPR)is a physical phenomenon.Due to the difference of light transmission characteristics between the different media,the light refraction and reflection will occur.Theoretically,the total internal reflection phenomenon will occur as long as the incidence angle is more than the critical angle(the critical angle is a specific incidence angle in case of the refraction angle is 90°).But the evanescent wave will decay the energy exponentially.If a thin layer of gold or silver films was painted onto the reflection interface,the charge oscillations generated by the free electrons of the metal surface activated by the incident light will be coupled with energy produced by the evanescent wave,specifically,is called SPR.Based on the SPR phenomenon,the SPR biosensor has emerged in 1980 s as the product of the combination of physics and biology.It has the capacity of testing the change of the molecular mass binding with the metal surfa ce of the biosensor itself by utilizing the reflection and refraction effects of the light between the two different media.In the past decade,the researches and application of SPR biosensor has been in progressing.As far the SPR biosensor has been used widely in various biomolecules‘ biological features such as lipids,proteins,nucleic acids,cells,bacteria,viruses and so on in view of the advantages including lable free,less sample consumption,real time and higher sensitivity.Rolling circle amplification(RCA)is develped on the basis of PCR.It is able to promote the amplification of DNA fragments in the condition of constant temperature.The basic theory of RCA was generated in 1990 s.At the beginning,the exo-minus Klenow and Bst DNA polymerases were employed in the experimental systems.Thanks to the higher capacity of constant amplification and chain replacement than the exo-minus Klenow and Bst DNA polymerases,the polymerase Phi 29 DNA started to be used in 1998.The template in the RCA system is the ring DNA molecules which can be complementarily combined with a short sequence,namely the primers.Under the effect of Phi 29 DNA,the RCA system can generate a number of long chain DNA fragments by means of amplification.Consequently,plenty of repeated DNA sequences will produced and the biological signals will amplified in a short time.RCA system excels at the real time detection and field testing because it does not need the temperature controling.In addition,the RCA system can prevent from the contamination due to the absence of the newborn single-stranded DNA(ss DNA)in the processing of DNA synthesis.The systematic evolution of ligands by exponential enrichment(SELEX)is a high-efficiency method which is generally introduced in the screening of the oligonucleotide fragments owning the high binding affinity with the target biomolecules.Nowadays the improvements on the basis of the basic SELEX system have significantly reduced the time costs,elevated the screening efficiency as well as decrease the screening costs.All the ssDNA and single-stranded RNA screened by means of the SELEX system are called aptamer,which have the ability to bind the targets specifically,i.e.the proteins,cells,bacteria,viruse and even the compounds.As a sort of innovative antibody,the aptamers have the potential in the application of the genetic treatment for the human cancers and the research fields of biomedicine.On the basis of previous studies in our laboratory,in the present study,by combining the liquid-phase RCA system and the SPR biosensor,we developed a new RCA amplification and testing system under the condition of the constant temperature based on the SPR biosensor.After the optimization of the entire system,the determination of the sensitivity and the reaction specificity of the system as a whole,we utilized the whole system to distinguish the MTBC and the MAC by means of the Padlock probe(PLP)designed from the pathogen relatively conservative internal transcribed space(ITS)model.Then,by the method of SELEX we screened the specific aptamer for Haemophilus influenzae.Meanwhile,we reduced the rate of nonspecific aptamer producing via adding the counter-selection in the screening processing.Furthermore,we analyzed secondary structure of the aptamers using software,testing the affinity and specificity and choose the highest affinity adapter involved in the detection of clinical samples.Materials and Methods1.Based on the previous research work in our laboratory,we further improved the various facilities of the SPR biosensor,including the hardware and software.Then we increased the number of the channels on the sensor chip surface,upgraded the temperature control system,improved the flow control system and optimized the self-assembly condition on the biosensor chip surface.2.We checked out the genetic information of the MTB in the G en Bank.Then we determined the ITS of 16s-23 s r RNA gene,designed the PLP,added the the restriction enzyme cutting site in the PLP,investigated the sensitivity and specificity of the reaction system and optimized the reaction conditions of the system.3.We established the real time detection approach targeting the DNA of the MTB by the SPR biosensor with the target-primed RCA.4.We screened the specific aptamers for the Haemophilus influenzae by means of the SELEX method.As the complex target,the Haemophilus influenzae cell body was employed to synthesis and build a random ssDNA library in vitro which had a length of 81 nt.The both ends of the library were constant and the database is about 1015 molecules.The sequence of the library was 5‘-C C C C T G C A G G T G A T T T T G C T C A A G T-(N 3 5)– A G T A T C G C T A A T C A G G C G G A T-3‘.The related primers were 5‘-C C C C T G C A G G T G A T T T T G C T C A A G T-3‘ and 5‘-A T C C G C C T G A T T A G C G A T A C T-3‘.A total of 12 rounds screening were made and the counterselection had been added in the 10-12 rounds.5.We predicted the secondary structure of the screened aptamers and determined their affinity and specificity.We selected one of the highest affinity for the clinical samples.After add the biotin to the aptamer,we immobilized the aptamer on the surface of the chip,then detected the clinical samples with the Haemophilus influenzae positive on the SPR biosensors.Results1.The improved SPR biosensor was more stable and precise.The temperature could be controled with an accuracy of ±0.1? in the range of 20-60?.Besides,the entire system was in negative pressure.The flow rate could be controled with an accuracy of ±1 ?roled in the range of 5-2000 ?l/min.The sensor chip had 8 channels which was the gold film prim at the size of 20mm×28.6mm and the thickness of 50 nm.The improved capture probes were immobilized on the surface of the biosensor via the thiol group modification and covalent coupling.2.The designed sequences of the probes according to the GenBank was as following: Target 1 of MTBC was 3'-C A C A C A A C C A C C G G T T G A A A C A A C A G T A C G T G G G C C G A G A G;Target 2 of MTBC was 3'-C A C A C A A C C A C C G G T T G A G A C A A C A G T A C G T G G G C C G A G A G;Target of MAC was 3'-C C T C C C T G A G G T G T G C C T G C C T G G C T C A C A A C A G A G T C C C G G;MTBC PLP: 3'-T T C A A C C G G T G G T A T C A T G A T T T T T A C G C G T C C A G C T A C G A C T C C A G C G T G T C T G T A T A T C T G G G C C C A C G T A C T G T T G T – P;MAC PLP: 3'-G C A C A C C T C A G G G A T C A T G A T T T T T A C G C G G C T A A T G G C C A G G C T A C G A C G T C T G T A T A T C T G T G T T G T G A G C C A G G C A G – P;The thiolated probe of MTBC: 3'– H S–(CH2)6–(T)20T C C A G C T A C G A C T C C A G C G T;The thiolated probe of MAC: 3'– H S–(CH2)6–(T)20G C T A A T G G C C A G G C T A C G A C.3.The SPR biosensor system with the liquid phase RCA targeted sequences amplification was established successfully.Both of the MTBC and MAC could be detected rapidly under the condition of the constant temperature.The new designed PLP made both of the RCA amplification and the restriction enzyme digestion be in processing simultaneously,which could be real time and online monitored.The lower limit of of the new SPR biosensor was MTBC 4.2x104 CFU/m L(0.005ng/?L)and MAC 3.7x104 CFU/m L(0.002ng/?L).4.A total of 6 aptamers were selected out via 12 rounds of SELEX screening.The sequences consistency of the 6 aptamers was 80.32% by means of the DNAMAN analysis.Three aptamers of them showed the OD values which were on behalf of the affinity between the aptamer itself and the Haemophilus influenzae and more than 1.5.Besides,the three aptamers above also had the high specificity reflected as the OD values of the affinity with counterselection were less than 0.5.5.Among the 12 cases of clinical samples of Haemophilus influenzae infection,11 cases were positive via the SPR biosensor detection.Conclusions1.The improved SPR biosensor performed very well,which could be benefit for the miniaturization and portability of the biosensor system in future.2.The PLP employed in the present study had high specificity.The RCA amplification could occur on the surface of the biosensor chip while the restriction enzyme cutting site was added,which promoted the rapid detection of the MTBC and the MAC by the SPR biosensor in the condition of the constant temperature.Therefore,the entire system could provide a brand new approach for the clinical diagnosis.3.In the present study,the aptamers which were specifically targeted to the Haemophilus influenzae and screened out via the SELEX could become a new way for the clinical detection. |
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