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Paper text:
long - term antibiotic abuse significantly increases the development of drug - resistant mutations in mycobacterium tuberculosis ( tb ) , especially point mutations .
numerous drug resistance - conferring point mutations in tb have been identified , and they include streptomycin - resistant mutations in the rpsl ( codons 43 and 88 ) or rrs genes , isonicotinic acid hydrazide - resistant mutations in the katg gene ( codons 315 and 463 ) , ethambutol - resistant mutations in the embb gene ( codons 285 , 303 , 306 , and 330 ) , and rifampin - resistant mutations in the ropb gene .
accurate and comprehensive detection of these point mutations by classical approaches is labor intensive and very difficult due to technical issues .
although bio - immobilization technologies such as the microarray and microbead assay permit high - throughput detection of these point mutations , the specificity and sensitivity of these assays to detect single - base mismatched target sequence by ordinary dna probes for hybridization remains a challenge [ 46 ] .
molecular beacons are a class of stem - loop dna probes with an internally quenched fluorophore and a quencher located at the ends of stem arms .
the probe binds to its specific target sequence , which induces a conformational change in the probe and dissociation of the fluorophore from the quencher , leading to restoration of fluorescence . because the loop is much longer than the stem , the perfect match between the loop and the target sequence determines the conformational change of the probe and disassociation of the stem .
well - designed molecular beacons with 1 mismatched base in their loops with the target sequences would not result in disassociation of the stem .
accordingly , molecular beacons offer many advantages over ordinary dna probes in detecting point mutations , such as higher sensitivity , specificity , and reusability ( figure 1 ) . at present ,
molecular beacons have been used for the detection of drug resistance - related point mutations in tb .
however , they are mainly used in non - immobilization assays , such as fluorescence - based quantitative pcr , which can not meet the requirements of high - throughput point mutation detection .
the combination of molecular beacons with bio - immobilization assays may be a promising strategy for the high - throughput detection of point mutations .
however , the difficulty in effectively immobilizing molecular beacons on the chip surface restricts the wide application of molecular beacons in bio - immobilization assays .
because the 2 ends of molecular beacons are occupied by a fluorophore and a quencher , molecular beacons can not be terminally modified for immobilization .
hence , many approaches have tried to modify the stem - arm by biotinylation , the most commonly used strategy ( figure 2a ) .
molecular beacons are attached on the chips through the biotin - avidin linkage , by which biotin is covalently attached to the stem arm , while avidin is covalently immobilized on the chip surface .
first , this approach usually causes a high background of fluorescent signal , which is a long - standing problem in on - chip fluorescence detection .
finally , the biotin - avidin linkage is not sequence - specific and not suitable for multiplex detection ( figure 2b , c ) .
although separation of immobilizing different probes on the chip surface before hybridization can allow multiple hybridizations simultaneously , this approach may cause low efficiency of hybridization because the efficiency of solid - liquid phase hybridization usually is lower than that of liquid - liquid phase hybridization ( figure 2d ) .
this way has no fluorescence fret phenomenon , for it is only modified by fluorophore without quencher , and the electrochemistry system is complicated and expensive .
paradoxically , high - efficiency liquid - liquid phase hybridization is one of the main strengths of molecular beacons .
however , little is known about whether it is possible to not only preserve the advantages of free molecular beacons in solution , but also to immobilize them on the chip surface for high - throughput detection of multiple molecules .
given the unique structural features of molecular beacons , we designed a novel asymmetric - loop molecular beacon in which the loop consists of 2 parts .
one ( segment a , adjacent to the fluorophore - labeled arm of the stem ) is complementary to a target and the other ( segment b , adjacent to the quencher - labeled arm of the stem ) is complementary to an oligonucleotide sequence immobilized on the chip surface ( figure 3a ) .
hence , the molecular beacon attaches to the chip surface through liquid - solid hybridization with the immobilized oligonucleotide sequence .
the segment a sequence in the loop of the molecular beacon can hybridize to its specific target simultaneously or sequentially .
because the loop of the molecular beacon contains 2 different segments of sequences , this probe is designated as the asymmetric - loop molecular beacon . in the absence of a target
, the stem hybrid will not dissociate and the probes will be washed off . when the novel probe encounters a target at the appropriate temperature , segment a of its loop hybridizes to the target sequence , leading to a conformational change of the probe . as a result ,
the stem hybrid of the probe dissociates to expose segment b , which in turn hybridizes to the oligonucleotide sequence immobilized on the chip surface , resulting in a fluorescent signal ( figure 3b ) .
because hybridizations occur in both the liquid - liquid ( segment a and targets ) and liquid - solid ( segment b and immobilized oligonucleotides ) phases in a single test , this novel assay is designated as a two - phase hybridization assay .
in this project , fam - dabclye will be tagged on the end of molecule beacon ( the maximum excitation wavelength of fam is 480 nm ) .
the main multiple drug resistance mutant gene points in mycobacterium tuberculosis include : streptomycin - resistant mutations in the rpsl gene ( codons 43 and 88 ) or rrs genes , isonicotinic acid hydrazide - resistant mutations in the katg gene ( codons 315 and 463 ) , ethambutol - resistant mutations in the embb gene ( codons 285 , 303 , 306 , and 330 ) , rifampin - resistant mutations in the ropb gene ( codons 526 , 531 ) , pyrazinamide - resistant mutations in the pnca gene , ethionamide - resistant mutations in the katg gene , fluoroquinolones - resistant mutations in the gyra gene ( codon 94 ) , and aminoglycosides - resistant mutations in 2 mutations ( a1400 g , a1401 g ) .
the main study process includes : 1 ) the above - mentioned main drug resistance points will be found in ncbi , and relative amplification primers will be designed and synthesized ; 2 ) asymmetric - loop molecular beacons and oligonucleotide probes aiming at these drug resistance mutant gene points will be individually designed and synthesized ; 3 ) microarray modified by oligonucleotide probes of the above points will be made and hybridization solution and instruments will be prepared ; 4 ) pcr of these points will be amplified , and pcr products will be purified ; 5 ) hybridization between pcr products and oligonucleotide probes on microarray will be completed ; 6 ) microarray eluted will be scanned by microarray scanner , and the results will be recorded and saved ; and 7 ) clinical drug resistance samples come from culture will be amplified by pcr and pcr products will be detected by microarray and compared with sequencing .
the asymmetric - loop molecular beacon - based two - phase hybridization assay is feasible because it has the following features .
first , asymmetric - loop molecular beacons retain the stem - loop structure of ordinary molecular beacons without requirement for further modification . in comparison with arm - modified molecular beacons ,
the synthesis of asymmetric - loop molecular beacons is easier , with little technical difficulty .
second , hybridization with asymmetric - loop molecular beacons has low background because only molecular beacons hybridizing to its target sequence can bind to oligonucleotides immobilized on chips , while free probes will be washed off .
thus the difference in the intensity between positive and negative fluorescent signals is huge , which should be much higher than that of biotin - avidin - based immobilized molecular beacons ( figure 3b ) .
furthermore , the sequence - specific hybridization between segment b of the probe and the immobilized oligonucleotides permits high - throughput detection of target molecules ( figure 3c ) .
in addition , the asymmetric - loop molecular beacon - based two - phase hybridization assay retains the advantages of liquid - liquid phase hybridization between probes and targets , which should be highly efficient .
finally , the asymmetric - loop molecular beacon - based two - phase hybridization assay may also be used in magnetic bead- and microbead - based assays ( figure 3d ) .
the asymmetric loop molecular beacon - based two - phase hybridization assay can overcome the difficulty of immobilizing molecular beacons in a relatively simple way .
this assay has the advantages of low fluorescence background , high - throughput detection , and high - efficiency hybridization , and holds great promise for accurate , comprehensive , and simultaneous detection of many drug resistance - related point mutations in tb ( figure 4 ) .
we propose a novel asymmetric - loop molecular beacon - based two - phase hybridization assay for accurate and high - throughput detection of multiple drug resistance - conferring point mutations . possessing the advantages of easy probe availability , multiplex detection , low background , and high - efficiency hybridization
, this novel assay may provide a new avenue for immobilization of molecular beacons and high - throughput detection of point mutations such as main multiple drug resistance mutant gene points in mycobacterium tuberculosis .
summarythe accurate and high - throughput detection of drug resistance - related multiple point mutations remains a challenge . 
 although the combination of molecular beacons with bio - immobilization technology , such as microarray , is promising , its application is difficult due to the ineffective immobilization of molecular beacons on the chip surface . here , we propose a novel asymmetric - loop molecular beacon in which the loop consists of 2 parts . 
 one is complementary to a target , while the other is complementary to an oligonucleotide probe immobilized on the chip surface . with this novel probe 
 , a two - phase hybridization assay can be used for simultaneously detecting multiple point mutations . 
 this assay will have advantages , such as easy probe availability , multiplex detection , low background , and high - efficiency hybridization , and may provide a new avenue for the immobilization of molecular beacons and high - throughput detection of point mutations .