An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents 英文参考文献.docVIP

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents 英文参考文献.doc

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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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