An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents 英文参考文献.docVIP
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An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents 英文参考文献
AnEfficientStrategyforBroad-RangeDetectionofLow
AbundanceBacteriawithoutDNADecontaminationof
PCRReagents
Shy-ShinChang1,2,3,Hsung-LingHsu4,Ju-ChienCheng5,Ching-PingTseng4,6*
1Graduate Institute of Clinical Medical Sciences, Department of Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, Republic of China,
2DepartmentofMedicine,CollegeofMedicine,ChangGungUniversity,Tao-Yuan,Taiwan,RepublicofChina,3DepartmentofFamilyMedicineandEmergencyMedicine,
ChangGungMemorialHospital,Tao-Yuan,Taiwan,RepublicofChina,4DepartmentofMedicalBiotechnologyandLaboratoryScience,ChangGungUniversity,Tao-Yuan,
Taiwan,RepublicofChina,5DepartmentofMedicalLaboratoryScienceandBiotechnology,ChinaMedicalUniversity,Taichung,RepublicofChina,6MolecularMedicine
ResearchCenter,ChangGungUniversity,Tao-Yuan,Taiwan,RepublicofChina
Abstract
Background:BacterialDNAcontaminationinPCRreagentshasbeenalongstandingproblemthathamperstheadoptionof
broad-rangePCRinclinicalandappliedmicrobiology,particularlyindetectionoflowabundancebacteria.Althoughseveral
DNAdecontaminationprotocolshavebeenreported,theyallsufferfromcompromisedPCRefficiencyordetectionlimits.To
date,nosatisfactorysolutionhasbeenfound.
Methodology/Principal Findings: We herein describe a method that solves this long standing problem by employing a
broad-rangeprimerextension-PCR(PE-PCR)strategythatobviatestheneedforDNAdecontamination.Inthismethod,we
firstdeviseafusionprobehavinga39-endcomplementarytothetemplatebacterialsequenceanda59-endnon-bacterial
tagsequence.WethenhybridizetheprobestotemplateDNA,carryoutprimerextensionandremovetheexcessprobes
usinganoptimizedenzymemixofKlenowDNApolymeraseandexonucleaseI.Thisstrategyallowsthetemplatestobe
distinguishedfromthePCRreagentcontaminantsandselectivelyamplifiedbyPCR.Toprovetheconcept,wespikedthe
PCRreagentswithStaphylococcusaureusgenomicDNAandappliedPE-PCRtoamplifytemplatebacterialDNA.Thespiking
DNA neither interfered with template DNA amplification nor caused false positive of the rea
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