Patent Application: US-201214385566-A

Abstract:
described according to an embodiment of the invention is a method and a system thereof , for the detection of at least one of staphylococcus aureus , the antibiotic resistant forms thereof , and the antibiotic resistant forms of other bacteria . the method comprises obtaining a sample , the extraction of nucleic acids from the sample and bringing the extracted nucleic acids into contact with a first primer pair complementary to a targeted segment of the attbscc gene , a second primer pair complementary to a targeted segment of the meca gene , and a third primer pair complementary to a targeted segment of the orfx gene .

Description:
reference will now be made in detail to particular aspects of a representative set of embodiments of the present disclosure , examples of which are illustrated in the accompanying drawings . while the disclosure will be described in conjunction with a number of embodiments , it will be understood that such embodiment ( s ) are not intended to limit the scope of the invention to the particular embodiments . on the contrary , the disclosure is intended to cover alternatives , modifications and equivalents , which may be included within the spirit and scope of the invention as defined by the appended claims . furthermore , in the following detailed description of embodiments in accordance with the present disclosure , numerous specific details ate set forth in order to provide a thorough understanding of the present invention . however , it will be recognized by one of ordinary skill in the art that the present invention may be practiced without these specific details . in other instances , well - known methods , procedures and components have not been described in detail as not to unnecessarily obscure aspects of embodiments of the present invention . staphylococcus aureus ( sa ) is a clinically significant pathogen that causes a wide spectrum of clinical manifestations , such as pneumonia , wound infections , septicemia and endocarditis . beta - lactam antimicrobial agents are often used as the preferred drags for serious sa infections . however , since the launch of methicillin in 1961 , methicillin - resistant staphylococcus aureus ( mrsa ) strains have evolved into notorious community pathogens known to cause serious hospital infections worldwide ( jevons , 1961 ). sa acquires methicillin resistance by insertion into the chromosome of a mobile generic element , staphylococcal cassette chromosomes ( sccs ). sccs are relatively large fragments of dna that always insert into the orfx gene on the sa chromosome . scc can encode antibiotic resistance and / or virulence determinants , including the methicillin resistance gene ( meca ). sccs can be classified into staphylococcal cassette chromosome mec ( sccmec ) or non - sccmec groups . the meca gene encodes as additional low - affinity penicillin - binding protein , pbp2a , which is not inhibited by existing beta - lactam antibiotics , i . e . resistance to b - lactam antibiotics is maintained by production of pbp2a , which fails to bind methicillin and other b - lactam antibiotics ( hiramatsu et al ., 2001 ). further , integration and excision of sccmec by recombinases occur within a specific attachment sits ( attbscc ) on the sa chromosome at the 3 ′ end of the orfx ( hiramatsu et al ., 2001 ). the present invention has been developed to provide a rapid and effective diagnosis of mrsa to facilitate or effectuate improved patient management . in addition to the more recent aforementioned assays for detection of mrsa , numerous molecular approaches which reduce the time for diagnosis of mrsa have been described in the following literature : baron , 1995 ; francois et al ., 2003 ; grisold et al ., 2002 ; jonas et al ., 2002 ; kearns et al ., 1999 ; reischl et al ., 2000 ; shresthat et al ., 2002 ; tan et al ., 2001 . disclosed herein are novel dna sequences relating to primers and probes which are specific to particular target regions within the attbscc , orfx and meca genes of sa . the attbscc site is found in orfx , and is a highly conserved region across sa strains ( hiramatsu , 2001 ). attbscc contains a 15 - bp sequence that , when sccmec is integrated in the chromosome , is present at the chromosome - sccmec junctions . thus , attbscc is a site that is potentially highly specific to mrsa but does not appear to be exploited in current assays for the detection of mrsa . other aspects of the present disclosure relate to further novel primers and probes derived from the novel dna sequences . also disclosed herein are processes for detecting the presence or absence of an mrsa strain in a sample comprising a mixed culture , such as a non - sterile specimen without a previous isolation , capture , or enrichment of one or more bacteria strains within . the dna sequences in the attbscc , meca and orfx genes of the sa genome were evaluated for suitable oligonucleotides for hybridization or pcr amplification by computer software analysis . potential candidate oligonucleotides for the primers and probes are evaluated and designed for specificity by blast analysis on the ncbi website , and for optimal sequence length using lasergene 7 software so as to prevent unwanted features such as secondary structures . a set of primers and probes for each of the 3 target regions attbscc , meca and orfx genes were designed to detect all 5 reported sub - types of mrsa namely nctc 10442 , n315 , 85 / 3907 , ca05 and wis , as shown in table 1 . “ y ”, “ r ” and “ w ” present in the primers and probes in table 1 each represents the degenerate bases which can be substituted with c or t ; a or g ; and a or t respectively . in some embodiments , the oligonucleotide sequences used for the primers and probes are as shown in table 1 , and listed as seq ids no . 1 - 10 in the sequence listing included herewith . in souse other embodiments , an oligonucleotide sequence comprising a fragment of at least 19 nucleotides of each of the corresponding primers and probes are used in the detection of the target regions attbscc , meca and orfx . each set comprising a forward primer , a reverse primer and a probe which are complementary to each of the meca and orfx sites are designed . in the case of the attbscc site , a set comprising a forward primer , two reverse primers and a probe which are complementary to the attbscc site are designed . the approximate amplicon size for each of the attbscc , meca and orfx target regions corresponding to each of the five reported mrsa sub - types are as shown in table 2 . three different fluorescent dyes are selected and each are used for the labelling of the probe targeting a specific region . each probe is labeled with a different fluorescent dye at the 5 ′ end . as each fluorescent dye emits fluorescence at a particular wavelength ( channel ), the simultaneous detection and differentiation of mrsa from other mr - microorganisms or non - methicillin resistant sa in a mixed culture sample is made possible . in some embodiments , dna is extracted from the sample supplied using the qiaamp ® dneasy blood and tissue mini kit , as per the manufacturer &# 39 ; s instructions . other non - limiting methods of dna extraction may also be used , as described in “ pcr protocols , a guide to methods and applications ” ( ed . innis , academic press , n . y . 1990 ) or any other dna extraction method generally practised by an ordinary person skilled in the art . in some embodiments , real - time pcr analysis is used to amplify the nucleic acids is the sample . multiplex real - time pcr analysis can be carried out according to the manufacturer &# 39 ; s instructions using the cfx96 real - time pcr detection system . in as embodiment , the specific pcr thermocycling conditions comprise 95 ° c . for 3 mins , followed by 35 cycles of 95 ° c . for 15 seconds and 60 ° c . for 30 seconds on the cfx96 real - time pcr detection system , of which a graphical representation of the above - mentioned protocol steps is shown in fig4 . the components of the real - time pcr set - up are as shows in table 3 : the detection of nucleic acids can be achieved via utilising a probe technology such as the taqman ® probe technology where the probe of the target region is labeled with a reporter dye and a quencher . in some embodiments , the attbscc probe is labeled with 6 - fam ( 520 nm ) at the 5 ′ end and black hole quencher ( bhq ) 1 at the 3 ′ end ; the meca probe is labeled with hex ( 556 nm ) at the 5 ′ end and bhq 1 at the 3 ′ end ; and the orfx probe is labeled with texas red ( 615 nm ) at the 5 ′ end and bhq 2 at the 3 ′ end , as shown in table 1 . in other embodiments , the attbscc probe , meca probe and orfx probe may each be labeled with a different fluorescent dye , i . e . 3 different fluorescent dyes , in which each of the fluorescent dye is compatible with the corresponding probe . upon exonuclease digestion during the pcr reaction cycles , the fluorescent dye is released from the quenched system and the fluorescence emitted is detected by the fluorimeter . in the embodiments where the attbscc , meca and orfx probes are labeled with the 6 - fam , hex and texas red fluorescent dyes respectively , the detection of the 6 - fam , hex and texas red fluorescences is at the 520 nm , 556 and 615 wavelengths respectively . in other embodiments , depending on the other types of fluorescent dyes which have been used , the detection of fluorescence emission is to be conducted in the corresponding wavelength channel indicated for the fluorescent dye , as per the specific manufacturer &# 39 ; s instructions . the type of combination of emission ( s ) detected at the different wavelengths of the fluorescent dye tagged to the specific probe will determine the type ( s ) of bacteria strain ( s ) present in the sample tested , as shown in tables 4a and 4b . the detection of fluorescence emission in all the 3 different wavelength channels will indicate the presence of mrsa in a mixed culture sample , in the absence of mrsa , the detection of an emission signal only in the 615 nm wavelength channel will indicate the presence of non - methicillin resistant sa , while the detection of an emission signal only in the 556 nm wavelength channel will indicate the presence of other methicillin resistant bacteria strains that are non - sa . table 5 as shown below , is a table of results from an embodiment of the pcr step of the method , and proves the effectiveness in the detection of mrsa in a known pure mrsa sample ( represented by a02 ) and known mixed culture samples of mrsa with sa , and mrsa with s . epidermidis ( represented by a03 and a04 respectively ). table 5 also shows the effectiveness in the detection of a known sample of pure sa that is non - methicillin resistant ( represented by a01 ) and the detection of a known sample of a bacteria strain that is methicillin resistant such as s . epidermidis ( represented by a06 ). in the case of a bacteria strain such as a known sample of e . coli ( represented by a05 ) which is neither methicillin resistant nor a sa strain , there is no signal in any of the wavelength channels . where “+” indicates emission will be detected for that bacteria strain as shown in fig1 , the primers and probe targeting the attbscc site in the multiplex assay are specific for detecting mrsa in the fluorescent channel 520 nm , in the presence of non - methicillin resistant sa , e . coli and s . epidermidis as controls . as shown in fig2 , the primers and probe targeting the meca site in the multiplex assay are specific for detecting mrsa in the fluorescent channel 556 nm , in the presence of non - methicillin resistant sa , e . coli and s . epidermidis as controls . as shown is fig3 , the primers and probe targeting the orfx site in the multiplex assay are specific for detecting sa in the fluorescent channel 615 nm , in the presence of e . coli and s . epidermidis as controls . ( 1 ) us food and ding administration . 510 ( k ) substantial equivalence determination decision summary assay and instrument combination template . retrieved from : http :// www . accessdata . fda . gov / cdrh_docs / reviews / k080837 . pdf ( 2 ) baron , e . j . 1995 . genetic aspects of methicillin resistance in staphylococcus aureus and methods used for its detection in clinical laboratories in the united states . j . chemother . 7 ( suppl . 3 ): 87 - 92 . ( 3 ) françois , p ., d . pittet , m . bento , b . pepey , p . vaudaux , d . lew , and j . schrenzel . 2003 . rapid detection of methicillin - resistant staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay . j . clin . microbiol . 41 : 254 - 260 . 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