We developed a high-throughput bead-based suspension array for simultaneous detection of 20 respiratory tract pathogens in clinical specimens. Pathogen-specific genes were amplified and hybridized to probes coupled to carboxyl-encoded microspheres. Fluorescence intensities generated via the binding of phycoerythrin-conjugated streptavidin with biotin-labeled targets were measured by the Luminex 100 bead-based suspension array system. The bead-based suspension array detected bacteria in a significantly higher number of samples compared to the conventional culture. There was no significant difference in the detection rate of atypical pathogensatypical pathogens or viruses between the bead-based suspension array and real-time PCR. This technology can play a significant role in screening patients with pneumonia.
TaubenbergerJKMorensDM. 1918 influenza: the mother of all pandemics. Emerg Infect Dis2006; 12: 15–22.
2.
CockburnWCDelonPJFerreiraW. Origin and progress of the 1968-69 Hong Kong influenza epidemic. Bull World Health Organ1969; 41: 345–8.
3.
GronebergDAZhangLWelteTZabelPChungKF. Severe acute respiratory syndrome: global initiatives for disease diagnosis. QJM2003; 96: 845–52.
4.
SullivanSJJacobsonRMDowdleWRPolandGA. 2009 H1N1 influenza. Mayo Clin Proc2010; 85: 64–76.
5.
MemishZA. MERS-CoV. An emerging viral zoonotic disease: three years after and counting. Recent Pat Antiinfect Drug Discov2014; 9: 159–60.
6.
KumarSWangLFanJKraftABoseMETiwariSVan DykeMHaigisRLuoTGhoshMTangHHaghniaMMatherELWeisburgWGHenricksonKJ. Detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-PCR assay with manual (enzyme hybridization) or automated (electronic microarray) detection. J Clin Microbiol2008; 46: 3063–72.
7.
LeeWMGrindleKPappasTMarshallDJMoserMJBeatyELShultPAPrudentJRGernJE. High-throughput, sensitive, and accurate multiplex PCR-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses. J Clin Microbiol2007; 45: 2626–34.
8.
KangYDengRWangCDengTPengPChengXWangGQianMGaoHHanBChenYHuYGengRHuCZhangWYangJWanHYuQWeiLLiJTianGWangQHuKWangSWangRDuJHeBMaJZhongXMuLCaiSZhuXXingWYuJDengMGaoZ. Etiologic diagnosis of lower respiratory tract bacterial infections using sputum samples and quantitative loop-mediated isothermal amplification. PLoS One2012; 7: e38743–e38743.
9.
WangDCoscoyLZylberbergMAvilaPCBousheyHAGanemDDeRisiJL. Microarray-based detection and genotyping of viral pathogens. Proc Natl Acad Sci USA2002; 99: 15687–92.
10.
BattagliaASchweighardtAJWallaceMM. Pathogen detection using a liquid array technology. J Forensic Sci2011; 56: 760–5.
11.
SchweighardtAJBattagliaAWallaceMM. Detection of anthrax and other pathogens using a unique liquid array technology. J Forensic Sci2014; 59: 15–33.
12.
HulseREKunklerPEFedynyshynJPKraigRP. Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue. J Neurosci Methods2004; 136: 87–98.
13.
MahonyJChongSMeranteFYaghoubianSSinhaTLisleCJaneczkoR. Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay. J Clin Microbiol2007; 45: 2965–70.
JeongESLeeKSHeoSHSeoJHChoiYK. Rapid identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae using triplex polymerase chain reaction in rodents. Exp Anim2013; 62: 35–40.
16.
SunKWangQHuangXHZhenMCLiWZhangLJ. Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers. Acta Pharmacol Sin2007; 28: 2011–8.
17.
MaassMKrauseEEngelPMKrugerS. Endovascular presence of Chlamydia pneumoniae in patients with hemodynamically effective carotid artery stenosis. Angiology1997; 48: 699–706.
18.
WangIKLaiHCYuCJLiangCCChangCTKuoHLYangYFLinCCLinHHLiuYLChangYCWuYYChenCHLiCYChuangFRHuangCCLinCHLinHC. Real-time PCR analysis of the intestinal microbiotas in peritoneal dialysis patients. Appl Environ Microbiol2012; 78: 1107–12.
19.
NomanpourBGhodousiABabaeiAAbtahiHTabriziMFeizabadiM. Rapid, cost-effective, sensitive and quantitative detection of Acinetobacter baumannii from pneumonia patients. Iran J Microbiol2011; 3: 162–9.
20.
LeeCSWetzelKBuckleyTWozniakDLeeJ. Rapid and sensitive detection of Pseudomonas aeruginosa in chlorinated water and aerosols targeting gyrB gene using real-time PCR. J Appl Microbiol2011; 111: 893–903.
21.
WhitbyPWCarterKBBurnsJLRoyallJALiPumaJJStullTL. Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR. J Clin Microbiol2000; 38: 4305–9.
22.
DunbarSA. Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clin Chim Acta2006; 363: 71–82.
23.
EarleyMCVogtRFJr.ShapiroHMMandyFFKellarKLBellisarioRPassKAMartiGEStewartCCHannonWH. Report from a workshop on multianalyte microsphere assays. Cytometry2002; 50: 239–42.
24.
DeregtDGilbertSADudasSPasickJBaxiSBurtonKMBaxiMK. A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses. J Virol Methods2006; 136: 17–23.
25.
TraczDMBackhousePGOlsonABMcCreaJKWalshJANgLKGilmourMW. Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus. J Med Microbiol2007; 56(Pt 1): 56–65.
26.
WallaceJWodaBAPihanG. Facile, comprehensive, high-throughput genotyping of human genital papillomaviruses using spectrally addressable liquid bead microarrays. J Mol Diagn2005; 7: 72–80.
27.
WilsonWJErlerAMNasarabadiSLSkowronskiEWImbroPM. A multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents. Mol Cell Probes2005; 19: 137–44.
28.
DunbarSAVander ZeeCAOliverKGKaremKLJacobsonJW. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system. J Microbiol Methods2003; 53: 245–52.
29.
LuJGetzGMiskaEAAlvarez-SaavedraELambJPeckDSweet-CorderoAEbertBLMakRHFerrandoAADowningJRJacksTHorvitzHRGolubTR. MicroRNA expression profiles classify human cancers. Nature2005; 435: 834–8.
30.
SpiroALoweMBrownD. A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry. Appl Environ Microbiol2000; 66: 4258–65.
31.
WangS. The microimmunofluorescence test for Chlamydia pneumoniae infection: technique and interpretation. J Infect Dis2000; 181(Suppl 3): S421–5.
32.
ChenYSWangDXLiHRChenYW. The applications of loop-mediated isothermal amplification in the detection of common pathogens causing lower respiratory tract infections. Chin J Tubercul Respir Dis2011; 17: 1–1.
33.
GandhiMKKhannaR. Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments. Lancet Infect Dis2004; 4: 725–38.
34.
RetalisPStrangeCHarleyR. The spectrum of adult adenovirus pneumonia. Chest1996; 109: 1656–7.
35.
KrunkoskyTMJordanJLChambersEKrauseDC. Mycoplasma pneumoniae host-pathogen studies in an air-liquid culture of differentiated human airway epithelial cells. Microb Pathog2007; 42: 98–103.
36.
HalbedelSStulkeJ. Tools for the genetic analysis of Mycoplasma. Int J Med Microbiol2007; 297: 37–44.
37.
SamransamruajkitRJitchaiwatSWachirapaesWDeerojanawongJSritippayawanSPrapphalN. Prevalence of Mycoplasma and Chlamydia pneumonia in severe community-acquired pneumonia among hospitalized children in Thailand. Jpn J Infect Dis2008; 61: 36–9.
38.
EunBWKimNHChoiEHLeeHJ. Mycoplasma pneumoniae in Korean children: the epidemiology of pneumonia over an 18-year period. J Infect2008; 56: 326–31.
39.
Reischl U, Lehn N, Simnacher U, Marre R, Essig A. Rapid and standardized detection of Chlamydia pneumoniae using LightCycler real-time fluorescence PCR. Eur J Clin Microbiol Infect Dis 2003;22:54–7.
40.
de RouxAEwigSGarcíaEMarcosMAMensaJLodeHTorresA. Mixed community-acquired pneumonia in hospitalised patients. Eur Respir J2006; 27: 795–800.
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