| BackgroundDue to the increase of population number and mobility, non-standard drug-use, as wellas the HIV/AIDS pandemic, a steady drug-resistant increase of M.tuberculosis hasthreatened tuberculosis (TB) control world-widely, despite advancements in the diagnosisand treatment of TB over the years. The multidrug-resistant strains were defined as beingresistant to at least isoniazid and rifampin. The emergence and spread of multidrug-resistantMycobacterium tuberculosis (MDRTB) even extensively drug resistant tuberculosis(XDRTB) posed an upward trend and represented an increasing public health problem.Conventional culture-based bacterial identification method or sequence analysis canprovide definitive results, however, it is time-consuming, laborious and expensive.Therefore, the development of simple, sensitive and low-cost method is important for therapid detection of drug-resistance. It is a timely and effective way to diagnose and manageTB patients, especially MDRTB infected.Because of high sensitivity of optical transducer, convenient operation and real-timemonitoring, surface plasmon resonance (SPR) biosensors have been adopted in numerousstudies on variety of bimolecular interaction including DNA, RNA, protein and bacteriumbased on different recognition elements. In a SPR biosensor assay, the change of therefractive index on the gold film correlates with the amount of analyte bound to the film. Inmost of SPR-based DNA detection with high sensitivity, nucleic acid amplification isadopted to amplify analyte mass, especially polymerase chain reaction (PCR). However, thenonspecific amplification from complicated thermal circling step, non-compatibility withon-chip amplification and increase of detection cost are still problems to be considered.Focusing on the development of isothermal amplification methods, RCA has shown itsattraction due to the specificity and multiplexing besides simplicity. The L-RCA by ligase relies on base pairing principle which requires perfect complementarity on the ligation nick.It not only forbids the mismatch but also has a low occurrence of false positive results whencompared to PCR. When used as a signal amplification method, this property enabled highmultiplexing without interference, direct amplification on solid phase and precise productlocalization. Moreover, RCA amplifies the circular PLP only, without accumulation oftarget templates over time, which minimizes the risk of contamination and the potentialbiohazard.In this study, we developed a SPR DNA biosensor method combined withsurface-anchored RCA to isothermally detect multiple point mutations associated withdrug-resistance in MDRTB. The Au nanoparticle signal amplification was adopted tofurther improve the sensitivity. The sensitivity and specificity of the SPR detection systemwere evaluated and the application was tested in real clinical samples. Besides, we alsodeveloped a SPR real-time detection method by target-primed RCA to discriminate TBbacilli from nontuberculous mycobacteria. This study lays the groundwork for applying theSPR biosensor for isothermal and rapid detection of resisitence associated mutations inM.tuberculosis.Methods1.The solid-phase RCA mode was contrasted by using magnetic beads. Theamino-modified oligonucleotide probe (capture probe) was immobilized on the surface ofthe carboxyl-modified magnetic beads through covalent coupling principle to form sensingmembrane. The optimum detection procedure and reaction condition were investigated. Theliquid phase RCA reaction condition was also investigated by the fluorescence quantitativemethod with SRBR Green II dye.2.Based on the previous study, the SPR biosensor platform was constructed withimprovements to increase the detection channel, improve temperature control, flow controland analyte circulating ways. The control software UMPHO-A600was designed tointerface with the SPR detector. The stability and reproducibility were evaluated. Theself-assembly of the sensor chip was improved to prepare microarrays.3.The sequences of five clinically meaningful mutations in MDRTB were searched forin the NCBI database. The padlock probe (PLP) containing a specific tag-sequence, ageneral sequence, and a target-specific region on each detection arm, was designed with Primer Premier5.0software. The capture probe containing tag-sequence with consistencyand specificity was designed by Array Designer4.0. The second structure prediction of thecircular PLP was also analyzed.4.Combination of Au nanoparticle signal amplification with the surface-anchored RCAresulted in cascade signal amplification on the chip surface. The experimental conditionsfor the detection system were also optimized. The synthetic oligonucleotides were used toevaluate the sensitivity and specificity of the detection system.5.According to the sequence of ITS in16s-23s rRNA gene found in GenBank, PLPwas designed to contain the restriction cutting site. The target-primed RCA was establishedto discriminate M.tuberculosis from NTM with the SPR biosensor. The dynamic real-timemonitoring of liquid-phase RCA with label-free manner was achieved.6.The detection system was demonstrated by targeting five resistance associatedmutations in MDRTB from34cased clinical samples. The sequencing was made asreference. The sensitivity and specificity of the developed SPR biosensor system formultiplex mutation detections of clinical samples were tested.Results1.The solid-phase RCA system was achieved on the carrier of magnetic beads and theamplification way was determined as ligation in solution combined with surfaceamplification. The optimum solid-phase RCA reaction condition was determined as0.5U/μL Phi29DNA polymerase，0.2μg/μL BSA，5%DMSO，1mM dNTPs. Theelectrophoresis verification showed that the circular PLP could be generated only in thepresence of mutant target to cause the following surface RCA reaction, whereas no reactionhappened.2.The sensor chip was the gold film prim with the size of20mm x28.6mm. Thedetection well was eight-channel tandem. The temperature was controlled with an accuracyof±0.1℃at25~60℃. The flow rate could be changed from5μL/min to2000μL/min withan accuracy of1μL/min. the detection well was in negative pressure to form a sealcondition to maintain the stability of the reaction during reaction. There was scarcelyinterference between channels to achieve the array detection. The thiol group modifiedcapture probes were self-assembled on the chip surface through the covalent binding withthe concentration of1μM. 3.The designed PLP can distinguish the target with single-base mismatch. RCA canamplify the SPR signal for almost10-fold. The positive mutation detection is achieved witha wild-type to mutant ratio of5000:1. The electrophoresis bands of ligation products, RCAproducts and restriction enzyme products of RCA were accurate and clear. The designedcapture probes with the similar length and Tm values (<1℃) can react under the samecondition, but also owned the high specificity. The optimum hybridization temperature inligation was45℃, while on the chip surface was37℃. The RCA duration time was30min.4.The probe sets were designed to target five common clinical resistance associatedmutation site, including isoniazid (INH): KatG315(AGCâ†'ACC), inhA-15(ACGâ†'ATG); rifampicin (PFP): rpoB526(CACâ†'TAC); ethambutol (EMB): embB306(ATGâ†'GTG); streptomycin (SM): rpsL43(AAGâ†'AGG). The multiplex amplification on chipsurface was achieved. The SPR signal difference between mutant and wild-type target wassignificant (p <0.01). The cross reaction result showed that the SPR signal differencebetween each mutation site was significant (p <0.01).5.The Au nanoparticle signal amplification system could effectively improve thesensitivity for2.1-fold, with15nm diameter, pH7.5and8%of concentration, whichshowed significant difference (p <0.01) compared to the negative control and blank control.The detection limit was5x10-12M synthetic oligonucleotides, and a linear correlation (y=615.29x+1323.8, R2=0.9846) was found within the concentration range of10-12M to10-8M.6.The developed target-primed RCA SPR biosensor system to isothermally (37℃)and rapidly (within4h) detect MTBC and MAC could be performed by only two steps. Theresults of clinical samples from SPR method and bacteria identification were consistence.The detection limit for MTBC was4.2X104CFU/mL (0.005ng/μL), and MAC was3.7x104CFU/mL (0.002ng/μL), respectively. The specially designed PLP could make theliquid-phase RCA and digestion reaction perform simultaneously, which achieve thedynamic real-time monitoring of RCA by SPR biosensor.7.The heated alkali lysis method was more simple, cost-effective and sensitive toextract genomic DNA in clinical samples. Compared to the sequencing method, thesensitivity and specificity of the SPR detection were92%and80%, respectively, for katG 315,100%and100%, respectively, for inhA-15,94.4%and100%, respectively, for rpoB526,95%and100%, respectively, for embB306,100%and100%, respectively, for rpsL43(Table2). According to the Kappa test,(p <0.05, Kappa>0.75) there exists significantconsistency between the two methods.Conclusions1.The developed isothermal amplification based SPR biosensor changed the detectionmethod of PCR-dependent traditional biosensors. The sealed and stable detection systemwith convenience and low power consumption was favor of miniaturization and portabilityapplication.2.The designed PLP had excellent capability of detecting the low-abundance mutationin the large background of wild targets, but also achieved the multiplex ligation with mixedtargets. The tag-sequence achieved the multiplex surface-anchored RCA with same reactioncondition and no interference, improved the multiplexing capabilities of the biosensor arrayand simplified the operation.3.Au nanoparticle signal amplification system could significantly increase the plasmonoscillations with a resonant frequency, thus improve the signal/noise ratio and thesensitivity. This system provided a reliable signal amplification method to detect traceanalyte, and hold great promise in scientific and clinical diagnostic application.4.The target-primed RCA SPR biosensor system achieved dynamically real-timemonitor the liquid amplification with label-free manners, which provided a rapid andisothermal detection method with specificity and sensitivity.5. Compared with the sequencing method, the developed SPR biosensor detectionsystem has good sensitivity and specificity in the detection of clinical samples, whichindicated that the novel SPR biosensor based on isothermal amplification would be apotential clinical diagnostic method for rapid detection of multiple resistance associatedmutation or other mutations associated disease in clinical samples... |
PDF Full Text Request