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1、JOURNAL OFCLINICALMICROBIOLOGY,0095-1137/00/$04.0010Feb.2000,p.781788Vol.38,No.2Copyright 2000,American Society for Microbiology.All Rights Reserved.Rapid Diagnosis of Bacteremia by Universal Amplification of23S Ribosomal DNA Followed by Hybridizationto an Oligonucleotide ArrayR.M.ANTHONY,T.J.BROWN,
2、ANDG.L.FRENCH*Department of Microbiology,Kings College St.Thomas Campus,St.Thomas Hospital,London SE1 7EH,United KingdomReceived 10 May 1999/Returned for modification 8 September 1999/Accepted 29 October 1999The rapid identification of bacteria in blood cultures and other clinical specimens is impor
3、tant for patientmanagement and antimicrobial therapy.We describe a rapid(4 h)detection and identification system thatuses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA,followed by reversehybridization of the products to a panel of oligonucleotides.This procedure w
4、as successful in discriminatinga range of bacteria in pure cultures.When this procedure was applied directly to 158 unselected positive bloodculture broths on the day when growth was detected,125(79.7%)were correctly identified,including 4 withmixed cultures.Nine(7.2%)yielded bacteria for which no o
5、ligonucleotide targets were present in the oligo-nucleotide panel,and 16 culture-positive broths(10.3%)produced no PCR product.In seven of the remainingeight broths,streptococci were identified but not subsequently grown,and one isolate of Staphylococcus aureuswas misidentified as a coagulase-negati
6、ve staphylococcus.The accuracy,range,and discriminatory power ofthe assay can be continually extended by adding further oligonucleotides to the panel without significantlyincreasing complexity or cost.The isolation of bacteria from blood cultures(bacteremia)isusually indicative of a serious invasive
7、 infection requiring ur-gent antimicrobial therapy.Different organisms have differentantimicrobial susceptibilities,and successful treatment is de-pendent on the prompt administration of the correct drug(6,14,23,25,26,33).Blood culture broths usually become posi-tive 8 to 24 h after inoculation.At t
8、his time,some indication ofbacterial identity can be obtained by Gram staining,but defin-itive identification and antibiotic susceptibilities are usually notavailable until 24 to 48 h later.This delay has two conse-quences:first,the patient may suffer if ineffective therapy isgiven for antibiotic-re
9、sistant organisms,and second,antibioticresistance may be encouraged if unnecessary antibiotics aregiven for sensitive organisms(3,21).Although in our hospitalnine bacterial groups(coagulase-negative staphylococci CoNS,Escherichia coli,Staphylococcus aureus,Pseudomonas aerugi-nosa,Enterococcus spp.,K
10、lebsiella spp.,Enterobacter spp.,Pro-teus spp.,and Streptococcus pneumoniae)account for morethan half of all clinically significant blood culture isolates,asmany as 50 species may be involved,and there are usually fewclinical clues as to the specific causative organism.Rapid spe-cies detection and i
11、dentification would facilitate earlier effec-tive therapy.Rapid diagnosis can be achieved by the direct detection ofcharacteristic bacterial genes in clinical specimens,and manyprimer sets have been developed to detect species-specificgenes in simple PCRs(10,18,31).These systems are usuallydesigned
12、to confirm the diagnosis of specific clinical syndromesand include the identification of Burkholderia pseudomallei inmelioidosis(9,34),S.pneumoniae in pneumococcal pneumo-nia and meningitis(7,22),Coxiella burnettii in Q fever(46),Listeria monocytogenes in listeriosis(5),Rhodococcus equi inrhodococco
13、sis in horses(42),Mycobacterium tuberculosis intuberculosis(12,36),Salmonella enterica serovar Typhi in ty-phoid fever(38,44),Mycoplasma pneumoniae in mycoplasmalpneumonia(30),Neisseria meningitidis in meningococcal men-ingitis(31),and Borrelia burgdorferi in Lyme disease(15).Sim-ilar test systems h
14、ave been designed for yeasts(29).In mostcases,the PCR has been performed on positive blood culturebottle samples,but some have been successful with directblood samples,serum,buffy coat specimens,or negative bloodculture bottle samples;these results suggest that DNA-basedmethods may be more sensitive
15、 than conventional bacteriologymethods.However,the use of different primers for different species isimpractical for the routine analysis of blood cultures that maycontain one or more of many possible pathogens.Either acomplex PCR with a mixture of large numbers of primers isneeded,or a large series
16、of individual PCRs must be run inparallel or sequentially.Multiplex PCR may be effective for alimited number of organisms,but as more primers are added,the sensitivity decreases and the chance that two unrelatedprimers will produce spurious products increases.Multiple in-dividual PCRs increase the e
17、xpense and complexity of theassay and,if they are run sequentially,the processing timeincreases for less common or unexpected pathogens.These problems can be avoided by using a single pair ofuniversal primers designed to amplify conserved stretches ofDNA from any bacterium present,followed by sequen
18、ce anal-ysis of the PCR product to determine the species.Previousinvestigators have usually chosen the 16S ribosomal DNA(rDNA)or the 16S-23S rDNA spacer region as a target foruniversal primers(35).The 16S rDNA is highly conserved,andsequences from it are now used in bacterial taxonomy(45).Incontrast
19、,the 16S-23S rDNA spacer region is highly variablewithin many species,frequently containing tRNA genes,andthis variation has been used for typing clinical isolates(2,18,41).There have been a few reports of the use of 16S rDNAvariation for the detection and identification of bacteria caus-*Correspond
20、ing author.Mailing address:Department of Microbi-ology,Kings College St.Thomas Campus,St.Thomas Hospital,London SE1 7EH,United Kingdom.Phone:44(0)171 922 8385.Fax:44(0)171 928 0730.E-mail:gary.frenchkcl.ac.uk.781ing bacteremia or meningitis(8,16)or for the differentiation ofbacteremia from other cau
21、ses of the sepsis syndrome(24).Detection of variation within fungal rDNA spacer regions byhybridization has been shown to be effective for the identifi-cation of yeast species in clinical specimens(43).Recently,sequence data for the large subunit(23S rDNA)have become available for a few bacterial sp
22、ecies.Analysis ofthese sequences(4,17,20,27,28,37)suggests that this regionshows more variation between species of medical importancethan 16S rDNA;therefore,universal primers designed to am-plify this region might be more useful for clinical diagnosis.These sequences can be analyzed by hybridizing t
23、he labeledPCR product to an array of oligonucleotides immobilized on asolid support(membrane or glass slides)or synthesized in situon silicon wafers(32).As both the target and the probe arepresent at much higher concentrations than is typical forSouthern blots,these hybridization reactions can be ca
24、rriedout in very short periods of time(less than 1 h).This methodis often referred to as reverse hybridization because the probesare immobilized and the target is in solution.Thus,organisms of bacteremia could be identified directlyfrom blood culture bottles by amplification of bacterial 23SrDNA,fol
25、lowed by reverse hybridization to an oligonucleotidearray designed to differentiate the sequence variation of thespecies.With this method,all specimens can be processed inthe same way,and an unlimited number of bacterial species orsequence variants can be incorporated without affecting thecomplexity
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