BACKGROUND
as a group of gram-positive bacteria, staphylococci strains are responsible for various tissues infection and a wide range of diseases, including skin infections, pneumonia, endocarditis, osteomyelitis, gastroenteritis, scalded skin syndrome and toxic shock syndrome  <cit> .
within species of staphylococci, staphylococcus aureus has been considered to be a major concern in both medicine and food safety  <cit> .
for other clinically significant staphylococcus strains, coagulase-negative staphylococci  strains have been reported as the most frequently isolated pathogens in intravascular catheter related infections  , thus become a frequent cause of nosocomial infection and bacteremia, especially in patients with indwelling medical devices  <cit> .
since the first report in  <dig>  methicillin-resistant staphylococcus aureus  has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world, which has raised a global challenge for clinicians, hospital epidemiologists and administrators  <cit> .
the meca gene, encoding the pbp2a protein and causing methicillin resistance in staphylococci, is located on staphylococcal cassette chromosome mec .
sccmec contains the mec gene complex  and the ccr gene complex   <cit> .
in recent years, mrsa strains have been considered to be a major example of the leading “super bugs”, with broad resistance to practically all β-lactam antibiotics and usually other multiple drugs due to the associated resistance genes carried by sccmec <cit> .
as consequence, the increasing awareness for the risk and hazard of mrsa strains raised the demands of an early, cost-effective, timely, specific and sensitive detection assay  <cit> .
as clinical mrsa diagnosis is concerned, staphylococcus strains have commonly been identified via routine standard procedures including colony morphology, gram staining, testing of catalase, hyaluronidase and coagulase, as well as the vitek  <dig> automated system and the api-staph commercial kit, which makes the detection of 16srrna somehow irrelevant.
however, despite the advantages of this assay, the further development and broad application of multiplex-pcr has been restricted by many factors, such as cross-reaction of different sets of primers, self-inhibition due to formation of dimmers, reduced amplification efficiency caused by the simultaneous and parallel amplification, undetectable influence of different targets, as well as the requirement for standard full use of external and internal quality control  for each assay.
as a novel analytical assay since the past decade, mass spectrometry has been applied for detection and diagnostics of various clinical microorganisms, with advantages on high-throughput process, sensitivity and specificity  <cit> .
however, this methodology requires trained personnel, operating space, complicated sample preparation procedure, as well as expensive equipment.
in the last decade, loop-mediated isothermal amplification  had been reported as a novel nucleic acid amplification method  <cit>   and applied to the detection of various pathogenic organisms, including escherichia coli o <dig> with the associated toxins stx <dig> and stx <dig> <cit> , yersinia pseudotuberculosis <cit> , salmonella <cit> , vibrio parahaemolyticus with the associated toxins tdh and trh <cit> , psuedomonas aeruginosa <cit>  and listeria monocytogenes <cit> .
the lamp methodology relies on an auto-cycling strand displacement dna synthesis performed by the bst dna polymerase large fragment under the isothermal conditions between 60-65°c, and the amplicons are mixtures of many different sizes of stem-loop dnas with several inverted repeats of the target sequence and cauliflower-like structures with multiple loops, which significantly simplify the reaction itself and the result determination.
the lamp assay includes  <dig> or  <dig> primers  to perform the amplification, and requires 45-60 min and 30-45 min without and with loop primers, respectively.
in comparision with pcr assay, the positive points of lamp method were simplicity, rapidity, specificity and sensitivity .
in the current study, a simple orfx-lamp assay had been developed, evaluated, optimized and further applied to detect a large scale of clinical mrsa strains, requiring approximately  <dig> mins for the whole process.
RESULTS
optimization of orfx-lamp assay
the orfx-lamp amplification had generated a large number of ladder-like pattern bands on agarose gel due to the characteristic structure up to the loading wells, with a bottom band of 212-bp size amplicon obtained by sequencing.
orfx-lamp assay had been performed under isothermal condition between 59°c and 66°c.
despite none of significant difference observed, the electrophoresis of lamp products which were amplified under 64°c exhibited slightly larger amount of dna amplicons when compared to other temperatures , which was consistent with studies previously.
as reaction length was concerned, various time points had been studied under 65°c and with 10 ng template dna, including 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min and 120 min.
for the lamp assay with loop primers, amplification had been initially detected at 30 min, and reached the maximal at 45 min .
nevertheless, without loop primers, amplification products could not be observed until 90 min.
all amplicons of orfx-lamp assay had been determined by electrophoresis, with observation directly by naked eye, as well as under uv light with sybr green dyed .
sensitivity and specificity of orfx-lamp assays
the detection limit of orfx-lamp assay had been studied by both minimal cfu of bacterial and template dna amount, which was found to be  <dig> copies of dna/tube and 10 cfu/reaction  , in comparision with the detection limit of pcr which was  <dig> copies of dna/tube and 103 cfu/reaction, indicating that the sensitive of lamp was 10- <dig> times sensitive than pcr.
as specificity was concerned, for primers, amplification could not be obtained in the absence of fip, bip, f <dig> or b <dig>  due to the indispensable role each of the primers plays in auto-cycling strand placement.
the lamp was only performed in the existence of both inner primers and outer primers.
in addition, for the orfx-lamp assay, high specificity was acquired when this assay was subjected to  <dig> reference strains, without any false positive amplification for non-mrsa strains .
orfx
-lamp
orfx
application of lamp assays on clinical mrsa strains
after establishment and optimization, orfx-lamp assay were applied to the detection of  <dig> clinical staphylococcus strains, including  <dig> mrsa,  <dig> mssa,  <dig> mrcns and  <dig> mscns strains , with comparative validation by standard pcr assay.
for application, rapid dna preparation process, simple heating equipments and results determination by observation directly by naked eye and under uv light had been applied.
as shown by the results, for total of  <dig> mrsa strains,  <dig>  and  <dig>  had been detected positive by lamp and pcr assays, respectively.
however, for other  <dig> non mrsa isolates, none and  <dig> yielded positive amplification.
the detection rate, positive predictive value  and negative predictive value  of orfx-lamp were  <dig> %, 100% and  <dig> %, respectively.
all reaction had been performed in triplicate to ensure reproducibility.
orfx
-lamp on clinical
staphylococcus
strains
samples, blood culture specimens included blood inoculated into aerobic and/or anaerobic blood culture medium.
tracheal secretion specimens included secretions from the trachea and bronchia and bronchoalveolar lavage fluid specimens.
wound specimens included surgical wound specimens but also specimens from ulcers, fistulae, abscesses, drainage fluids, catheters sites, percutaneous endoscopic gastrostomy insertion sites, and tracheostomas.
urine contained native urine from urinary catheters or bladder puncture and inoculated culture systems.
other specimens consisted of skin swabs specimens , as well as swab specimens of the throat, tonsils, eye, ear, and vagina; body liquids; puncture exudates.
discussion
as one of the globally widespread clinical pathogens, mrsa strains have attained a heightened concern for its rapid and accurate detection, which has become a common issue in the diagnostics of mrsa.
current mrsa diagnosis methodologies include routine standard procedures , vitek  <dig> automated system, api-staph kit, immunological assays, mass spectrometry, pcr , etc.
mrsa strains exhibit resistance to practically all β-lactam antibiotics and commonly other drugs due to the meca and other resistance genes carried by sccmec.
however, for type i  and iv  sccmec , the meci-encoded repressor function may lead to the low-level methicillin resistance.
in the case of type ii  and iii  sccmec , the is431-mediated deletion of meci and further derepression of meca transcription may cause the expression of methicillin resistance.
as consequence, differing from biochemical methodologies, the accurate diagnostic identification of mrsa relies on the detection of highly specific and conserved targets within sccmec.
as orfx was considered to be highly specific target for both s. aureus and sccmec, a distinctive mrsa detection methodology based on orfx-lamp was developed in the present study.
in addition, a number of attributes, including rapidity, simplicity in operation, specificity, sensitivity and expense, were also evaluated to verify the application of the orfx-lamp assay for mrsa detection.
targeting on six distinct regions, inner and outer primers of the lamp assay were highly specific in comparison with conventional pcr techniques.
in this study, the specificity of the orfx-lamp assay was verified by indispensibility of each primer, without any false positive test obtained from reference strains and 100% ppv for application.
as sensitivity was concerned, in comparison with regular pcr   <cit>  and previous lamp detection methodoloy   <cit> , the detection limit of orfx-lamp was found to be  <dig> copies dna/tube and 10 cfu/reaction.
for rapidity, approximate 60 min was required in the application of orfx-lamp, including dna process, isothermal reaction and result determination.
CONCLUSIONS
the first study on detection of mrsa by lamp assay was reported lately  <cit> , however,  <dig> individual lamp assays with targets including 16srrna, fema and meca were laborious and time and expense demanding.
also, in another preliminary staphylococcus lamp identification assay, 120 min and  <dig> targets  were required for the procedure  <cit> .
in this current study, a simple lamp assay targeting on orfx for rapid mrsa detection had been developed, evaluated and optimized, which required approximately 60 min for each test and used the highly conserved target orfx.
this orfx-lamp had been further applied to the detection of  <dig> clinical staphylococcus strains, and the sensitivity, specificity, ppv and npv were found to be  <dig> %, 100%, 100% and  <dig> %, respectively.
in comparison with conventional pcr, this established lamp assay exhibited advantages on detection limit, sensitivity, simplicity and rapidity.
in conclusion, this orfx-lamp assay had been demonstrated to be a valid and rapid detection method on mrsa, which will undoubtedly aid in the broad application of bacteriological mrsa detection.

 as a group of gram-positive bacteria, staphylococci strains are responsible for various tissues infection and a wide range of diseases, including skin infections, pneumonia, endocarditis, osteomyelitis, gastroenteritis, scalded skin syndrome and toxic shock syndrome. within species of staphylococci, staphylococcus aureus has been considered to be a major concern in both medicine and food safety. for other clinically significant staphylococcus strains, coagulase-negative staphylococci strains have been reported as the most frequently isolated pathogens in intravascular catheter related infections, thus become a frequent cause of nosocomial infection and bacteremia, especially in patients with indwelling medical devices. since the first report in methicillin-resistant staphylococcus aureus has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world, which has raised a global challenge for clinicians, hospital epidemiologists and administrators. the meca gene, encoding the pbp2a protein and causing methicillin resistance in staphylococci, is located on staphylococcal cassette chromosome mec. sccmec contains the mec gene complex and the ccr gene complex. in recent years, mrsa strains have been considered to be a major example of the leading “super bugs”, with broad resistance to practically all β-lactam antibiotics and usually other multiple drugs due to the associated resistance genes carried by sccmec. as consequence, the increasing awareness for the risk and hazard of mrsa strains raised the demands of an early, cost-effective, timely, specific and sensitive detection assay. as clinical mrsa diagnosis is concerned, staphylococcus strains have commonly been identified via routine standard procedures including colony morphology, gram staining, testing of catalase, hyaluronidase and coagulase, as well as the vitek automated system and the api-staph commercial kit, which makes the detection of 16srrna somehow irrelevant. however, despite the advantages of this assay, the further development and broad application of multiplex-pcr has been restricted by many factors, such as cross-reaction of different sets of primers, self-inhibition due to formation of dimmers, reduced amplification efficiency caused by the simultaneous and parallel amplification, undetectable influence of different targets, as well as the requirement for standard full use of external and internal quality control for each assay. as a novel analytical assay since the past decade, mass spectrometry has been applied for detection and diagnostics of various clinical microorganisms, with advantages on high-throughput process, sensitivity and specificity. however, this methodology requires trained personnel, operating space, complicated sample preparation procedure, as well as expensive equipment. in the last decade, loop-mediated isothermal amplification had been reported as a novel nucleic acid amplification method and applied to the detection of various pathogenic organisms, including escherichia coli o with the associated toxins stx and stx, yersinia pseudotuberculosis, salmonella, vibrio parahaemolyticus with the associated toxins tdh and trh, psuedomonas aeruginosa and listeria monocytogenes. the lamp methodology relies on an auto-cycling strand displacement dna synthesis performed by the bst dna polymerase large fragment under the isothermal conditions between 60-65°c, and the amplicons are mixtures of many different sizes of stem-loop dnas with several inverted repeats of the target sequence and cauliflower-like structures with multiple loops, which significantly simplify the reaction itself and the result determination. the lamp assay includes or primers to perform the amplification, and requires 45-60 min and 30-45 min without and with loop primers, respectively. in comparision with pcr assay, the positive points of lamp method were simplicity, rapidity, specificity and sensitivity. in the current study, a simple orfx-lamp assay had been developed, evaluated, optimized and further applied to detect a large scale of clinical mrsa strains, requiring approximately mins for the whole process.  optimization of orfx-lamp assay the orfx-lamp amplification had generated a large number of ladder-like pattern bands on agarose gel due to the characteristic structure up to the loading wells, with a bottom band of 212-bp size amplicon obtained by sequencing. orfx-lamp assay had been performed under isothermal condition between 59°c and 66°c. despite none of significant difference observed, the electrophoresis of lamp products which were amplified under 64°c exhibited slightly larger amount of dna amplicons when compared to other temperatures, which was consistent with studies previously. as reaction length was concerned, various time points had been studied under 65°c and with 10 ng template dna, including 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min and 120 min. for the lamp assay with loop primers, amplification had been initially detected at 30 min, and reached the maximal at 45 min. nevertheless, without loop primers, amplification products could not be observed until 90 min. all amplicons of orfx-lamp assay had been determined by electrophoresis, with observation directly by naked eye, as well as under uv light with sybr green dyed. sensitivity and specificity of orfx-lamp assays the detection limit of orfx-lamp assay had been studied by both minimal cfu of bacterial and template dna amount, which was found to be copies of dna/tube and 10 cfu/reaction, in comparision with the detection limit of pcr which was copies of dna/tube and 103 cfu/reaction, indicating that the sensitive of lamp was 10- times sensitive than pcr. as specificity was concerned, for primers, amplification could not be obtained in the absence of fip, bip, f or b due to the indispensable role each of the primers plays in auto-cycling strand placement. the lamp was only performed in the existence of both inner primers and outer primers. in addition, for the orfx-lamp assay, high specificity was acquired when this assay was subjected to reference strains, without any false positive amplification for non-mrsa strains. orfx -lamp orfx application of lamp assays on clinical mrsa strains after establishment and optimization, orfx-lamp assay were applied to the detection of clinical staphylococcus strains, including mrsa, mssa, mrcns and mscns strains, with comparative validation by standard pcr assay. for application, rapid dna preparation process, simple heating equipments and results determination by observation directly by naked eye and under uv light had been applied. as shown by the results, for total of mrsa strains, and had been detected positive by lamp and pcr assays, respectively. however, for other non mrsa isolates, none and yielded positive amplification. the detection rate, positive predictive value and negative predictive value of orfx-lamp were %, 100% and %, respectively. all reaction had been performed in triplicate to ensure reproducibility. orfx -lamp on clinical staphylococcus strains samples, blood culture specimens included blood inoculated into aerobic and/or anaerobic blood culture medium. tracheal secretion specimens included secretions from the trachea and bronchia and bronchoalveolar lavage fluid specimens. wound specimens included surgical wound specimens but also specimens from ulcers, fistulae, abscesses, drainage fluids, catheters sites, percutaneous endoscopic gastrostomy insertion sites, and tracheostomas. urine contained native urine from urinary catheters or bladder puncture and inoculated culture systems. other specimens consisted of skin swabs specimens, as well as swab specimens of the throat, tonsils, eye, ear, and vagina; body liquids; puncture exudates. discussion as one of the globally widespread clinical pathogens, mrsa strains have attained a heightened concern for its rapid and accurate detection, which has become a common issue in the diagnostics of mrsa. current mrsa diagnosis methodologies include routine standard procedures, vitek automated system, api-staph kit, immunological assays, mass spectrometry, pcr, etc. mrsa strains exhibit resistance to practically all β-lactam antibiotics and commonly other drugs due to the meca and other resistance genes carried by sccmec. however, for type i and iv sccmec, the meci-encoded repressor function may lead to the low-level methicillin resistance. in the case of type ii and iii sccmec, the is431-mediated deletion of meci and further derepression of meca transcription may cause the expression of methicillin resistance. as consequence, differing from biochemical methodologies, the accurate diagnostic identification of mrsa relies on the detection of highly specific and conserved targets within sccmec. as orfx was considered to be highly specific target for both s. aureus and sccmec, a distinctive mrsa detection methodology based on orfx-lamp was developed in the present study. in addition, a number of attributes, including rapidity, simplicity in operation, specificity, sensitivity and expense, were also evaluated to verify the application of the orfx-lamp assay for mrsa detection. targeting on six distinct regions, inner and outer primers of the lamp assay were highly specific in comparison with conventional pcr techniques. in this study, the specificity of the orfx-lamp assay was verified by indispensibility of each primer, without any false positive test obtained from reference strains and 100% ppv for application. as sensitivity was concerned, in comparison with regular pcr and previous lamp detection methodoloy, the detection limit of orfx-lamp was found to be copies dna/tube and 10 cfu/reaction. for rapidity, approximate 60 min was required in the application of orfx-lamp, including dna process, isothermal reaction and result determination.  the first study on detection of mrsa by lamp assay was reported lately, however, individual lamp assays with targets including 16srrna, fema and meca were laborious and time and expense demanding. also, in another preliminary staphylococcus lamp identification assay, 120 min and targets were required for the procedure. in this current study, a simple lamp assay targeting on orfx for rapid mrsa detection had been developed, evaluated and optimized, which required approximately 60 min for each test and used the highly conserved target orfx. this orfx-lamp had been further applied to the detection of clinical staphylococcus strains, and the sensitivity, specificity, ppv and npv were found to be %, 100%, 100% and %, respectively. in comparison with conventional pcr, this established lamp assay exhibited advantages on detection limit, sensitivity, simplicity and rapidity. in conclusion, this orfx-lamp assay had been demonstrated to be a valid and rapid detection method on mrsa, which will undoubtedly aid in the broad application of bacteriological mrsa detection. 
as clinical mrsa diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for mrsa detection. for application, and tested strains had been detected positive by lamp and pcr assays. high specificity was observed when orfx-lamp assay was subjected to reference strains. the detection rate, positive predictive value and negative predictive value of orfx-lamp were %, 100% and % respectively. the 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be dna copies and 10 cfu/reaction. the established orfx-lamp assay had been demonstrated to be a valid and rapid detection method on mrsa. the protocol was designed by targeting orfx, a highly conserved open reading frame in s. aureus. one hundred and sixteen reference strains, including gram-positive and gram-negative isolates, were included for evaluation and optimization of the orfx-lamp assay. this assay had been further performed on staphylococcus strains and were comparatively validated by pcr assay using primers f and b with rapid template dna processing, simple equipments and direct result determination applied. methicillin-resistant staphylococcus aureus has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world.
 methicillin-resistant staphylococcus aureus has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. as clinical mrsa diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for mrsa detection. this study aimed at developing a simple loop-mediated isothermal amplification assay targeting on orfx for the rapid detection of methicillin-resistance staphylococcus aureus.  the protocol was designed by targeting orfx, a highly conserved open reading frame in s. aureus. one hundred and sixteen reference strains, including gram-positive and gram-negative isolates, were included for evaluation and optimization of the orfx-lamp assay. this assay had been further performed on staphylococcus strains and were comparatively validated by pcr assay using primers f and b with rapid template dna processing, simple equipments and direct result determination applied. the indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°c for 45 min. the 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be dna copies and 10 cfu/reaction. high specificity was observed when orfx-lamp assay was subjected to reference strains. for application, and tested strains had been detected positive by lamp and pcr assays. the detection rate, positive predictive value and negative predictive value of orfx-lamp were %, 100% and % respectively.  the established orfx-lamp assay had been demonstrated to be a valid and rapid detection method on mrsa. loop-mediated isothermal amplification mrsaorfx 