简介：Objective:Toinvestigatetheapplicationofpolymerasechainreaction(PCR)detectionofHaemophilusducreyiinclinicaldiagnosisofchancroid.Methods:Nucleotidesequencesof16srRNAgenespecificforH.dureyiwereusedtodevelopprimersetsforamplificationoftwostrains.TheamplifiedproductsweretestedviaPCRandsequencedbyelectrophoresisina1.5%gel.TheseproductswerecomparedwiththoseofheterogeneousspeciesorrelatedbacteriatotestthespecificityofthePCRassay.PCRamplificationwithdifferentconcentrationsofH.ducreyiwasperformedtotestitssensitivity.Results:PCRamplificationoftwostrainsofH.ducreyiproducedasinglebandofexpected438bplength.ThesequencewasidentifiedwithgenomicDNA.Noneoftheother19referencespeciesamplifiedunderthesameconditionsgavethisresult.ThehighestsensitivityofPCRassayinthepresenttestwas10ng/L.Conclusions:PCRassayfordetectionofH.ducreyiisarapid,specific,andsensitivedetectionmethod.Iflaboratoryconditionsarestrictlycontrolled,PCRassayisapotentiallyusefullaboratorytestforH.ducreyiinfectiondiagnosis.

简介：Objective:Todetectandquantitategenitalherpessimplexvirus(HSV)DNAinspecimensfrom100patientsclinicallydiagnosedwithgenitalherpes.Methods:PolymeraseChainReaction(PCR)andenzyme-linkedimmunosorbentassay(ELISA)wereusedwithastandardcurveofDNAcopiesofHSVasquantitativecontrast.Results:Ninety-threecaseswereconfirmedHSVpositiveand7caseswerefoundtobenegative.Therewere58casesofHSV-2(62.4%)and35casesofHSV-1(37.6%)amongthe93positivecases.ThenumberofDNAplasmidsrangedfrom115to1.1×l0^5per250pLamongthe93positivesamples(mean=7.1×10^4/250μL).ThenumberofHSVDNAplasmidsrangedfrom136to1.1×l0^5copiesper250pL(mean=7.6×10^4)amongthosewithHSV-2,and115to9.4×10^4per250pL(mean=6.3×10^4)amongthosewithHSV-1.Meanwhile10μLofextractedanddissolvedDNArandomlytakenfrom8eachofHSV-2andHSV-1samplesweretested.ThenumberofHSV-2DNAplasmidsrangedfrom35copiesto2.7×10^4(Mean=l.8×10^4)andthenumberofHSV-1DNArangedfrom29to2.5×10^4(Mean=1.6×10^4).Inthe7negativecases,thequantityofHSVplasmidswaszero.Conclusion:ThesensitivityofELISAquantitation(93%)isequaltothatofSouthernblot.ThesensitivityofPCRfordiagnosisis91%,and88%forPCRtyping.

简介：

简介：Objectives:Todevelopamulti-nestedpolymerasechainreactioninanassaytodetectearlyTreponemapallidumandHaemophilusducreyiDNAintheswabsofgenitalulcers.Methods:Fourpairsofouterandinnerprimers,specifictothebasicmembraneproteingeneofTreponemapallidumandtothe16srRNAgeneofHducreyiweresynthesized.Themulti-nestedPCRwasdevelopedandappliedtodetectTreponemapallidumandHaemophilusdicreyiinclinicalswabs.Result:ThetwosamplesofstandardstrainsofHaemophilusducreyiandoneTreponemapallidumwereamplifiedandshowed309-bprRNAgeneofHaemophilusducreyiand506-bpDNAofTreponemapalidum,respectively.Outof51samplesofgenitalulcerdetected,29showedTreponemapallidumpositiveproductandnoHaemophilusducreyiDNAwasfound.Conclusion:Themulti-nestedPCRforTreponemapallidumandHaemophilusducreyicouldbeusefulforearlydetectionanddistinguishingdiagnosisbetweensyphilisandchancroid.

简介：Objectives:Toevaluatetheefficacyofnestedpolymerasechainreaction(PCR)withfirstvoidurine(FVU)forthediagnosisofMycoplasmahominisinmalepatients.Methods:MatchedFVUspecimensandurethralswabswerecollectedfrom194malepatientswithNongonococcalUrethritisandtestedbynestedPCRandcellculture.Cellculturewasusedasagoldstandardforevaluatingotherassaytechniques.Results:ForFVUnestedPCRassayandFVUcellculture,ourresultsshowedthatthesensitivitywas100%and93.3%;specificitywas97.0%and98.2%;positivepredictivevalue(PPV)was85.7%and90.3%,negativepredictivevalue(NPV)was100%and98.8%,respectively.Thetotalconsistencybetweenthetwotechniqueswas97.4%.Conclusions:ForthediagnosisofMycoplasmahominisinmen,nestedPCRdetectingFVUisahighlysensitiveandspecificmethod.FirstvoidurinecanreplaceswabcultureorPCRintermsofacceptabilityandfeasibility.

简介：Objective:ToevaluatetheclinicalapplicationofmultiplexPCRinthedetectionofTreponemapallidum,Herpessimplexvirus(HSV),andHaemophilusducreyi.Method:ThreestandardstrainswereusedtosetupamultiplexPCR(MPCR)fordetectingsyphilis,herpesgenitalis,andchancroidsimultaneously.Samplesfrom122patientswithgenitalulcerdisease(GUD)weresubjectedtoMPCRandtheresultswerecomparedwiththeseofdark-fiddmicroscopyandTPserology,HSVanligenELISA,andH.ducreyiculture,Result:Inthe122patientswithGUD,MPCRidentified34casesofT.palliduminfection,40casesofHSVinfection,and2casesofmixedinfectionofT.pallidumandherpes.NopositiveresultsofH.ducreyiwerefound.ThesensitivityofMPCRtoT.pallidumandherpeswas100%and93.3%,respectivdy.Thesensitivitiesofdark-fieldmicroscopyandTPserology,HSVantigenELISA,andH.ducreyiculturewas35.3%,50%and100%,respectively.Conclusion:MPCRshowedarelativelyhighersensitivityforT.pallidumascomparedwiththeroutinetechniques.AlthoughitssensitivityforHSVwasnotasgoodasthatofantigenELISA,italsoyieldedahighdetectionrate.MPCRcandetectmorethanonepathogen.Itissimple,quick,sensitive,andsuitableforclinicaluseorepidemiologicalinvestigation.

简介：Objective:TostudytheprevalenceofTrichomonasvaginalis(TV)infectioninChinesemalepatientswithnongonococcalurethritis(NGU),toevaluatethesensitivityandspecificityofurine-basedandurethralswabpolymerasechainreaction(PCR)detection,tosetupamethodfornon-invasivedetectionofmaleTVinfection.Method:OnehundredandfivemaleNGUpatientswereselectedfromaBeijingSTDclinic.Twourethralswabswereobtainedfromeachpatient,onefortheInPouchTVculturesystemandtheotherforPCR.Inaddition,onefirstvoidurinespecimenwascollectedforPCRdetection.Culturewasconsideredthe“goldstandard”.Thesensitivity,specificity,positivepredictivevalue(PPV)andnegativepredictivevalue(NPV)ofthetwoPCRdetectionswerecomparedtocultureresults.Results:Theprevalenceofurine-basedPCRandurethralswabPCRdetectionwas3.81%(4/105)and4.76%(5/105)respectively.Comparedtoculture,thesensitivity,specificity,PPVandNPVwere80%,100%,100%and99%forurine-basedPCRand80%,99%,80%and99%forurethralswabPCR.Conclusion:TVisoneoftheetiologicalagentsinmaleNGU,witha4.76%prevalenceofinfectioninourstudy.TheurinebasedPCRdetectionhashighersensitivityandspecificityandprovidesanoninvasivemethodmorefeasibleinpractice.

简介：AbstractBackground:Rapid and accurate detection of drug resistance in Mycobacterium tuberculosis is critical for effective control of tuberculosis (TB). Herein, we established a novel, low cost strategy having high accuracy and speed for the detection of M. tuberculosis drug resistance, using gene splicing by overlap extension PCR (SOE PCR).Methods:The SOE PCR assay and Sanger sequencing are designed and constructed to detect mutations of rpoB, embB, katG, and inhA promoter, which have been considered as the major contributors to rifampicin (RFP), isoniazid (INH), and ethambutol (EMB) resistance in M. tuberculosis. One hundred and eight M. tuberculosis isolates came from mycobacterial cultures of TB cases at Chongqing Public Health Medical Center in China from December 2018 to April 2019, of which 56 isolates were tested with the GeneXpert MTB/RIF assay. Performance evaluation of the SOE PCR technique was compared with traditional mycobacterial culture and drug susceptibility testing (DST) or GeneXpert MTB/RIF among these isolates. Kappa identity test was used to analyze the consistency of the different diagnostic methods.Results:We found that the mutations of S531L, S315T and M306V were most prevalent for RFP, INH and EMB resistance, respectively, in the 108 M. tuberculosis isolates. Compared with phenotypic DST, the sensitivity and specificity of the SOE PCR assay for resistance detection were 100.00% and 88.00% for RFP, 94.64% and 94.23% for INH, and 68.97% and 79.75% for EMB, respectively. Compared with the GeneXpert MTB/RIF, the SOE PCR method was completely consistent with results of the GeneXpert MTB/RIF, with a concordance of 100% for resistance to RFP.Conclusions:In present study, a novel SOE PCR diagnostic method was successfully developed for the accurate detection of M. tuberculosis drug resistance. Our results using this method have a high consistency with that of traditional phenotypic DST or GeneXpert MTB/RIF, and SOE PCR testing in clinical isolates can also be conducted rapidly and simultaneously for detection of drug resistance to RFP, EMB, and INH.

简介：无

简介：AbstractObjective:Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification data. Recently, several strategies have been utilized for validating reference genes in different human tissues. However, no universal reference genes have been described that accurately summarize transcriptional activity in human spermatozoa.Methods:Using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy rates. We assessed the stability of reference genes using three different algorithms, namely geNorm, NormFinder, and BestKeeper. We then identified the most stable reference genes.Results:Male-enhanced antigen 1 (MEA1) was identified as the most stably expressed reference gene, followed by testis-enhanced gene transcript (TEGT).Conclusions:We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.

简介：无

简介：AbstractBackground:Patients carrying the HongKongαα (HKαα) allele and -α3.7/αααanti-4.2 could be misdiagnosed as -α3.7/αα by the current conventional thalassemia detection methods, leading to inaccurate genetic counseling and an incorrect prenatal diagnosis. This study was aimed to accurately analyze the genotypes of HKαα carriers and -α3.7/αααanti-4.2.Methods:Samples were collected in our hospital from July 2017 to October 2019. Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction (Gap-PCR) and reverse dot blot (RDB). Anti-4.2 multiplex-PCR was used to confirm carriers of the αααanti-4.2 duplication with -α3.7 deletion. Two-round nested PCR and multiplex ligation-dependent probe amplification (MLPA) were applied to accurately identify and confirm their genotypes. For data analysis, we used descriptive statistics and Fisher’s exact tests.Results:Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples. The results showed that α, β, and αβ compound thalassemia were identified in 1190 (46.78%), 1286 (50.55%), and 68 (2.67%) cases, respectively. A total of 227 samples from thalassemia patients were identified as -α3.7/αα by Gap-PCR, and the genotypes of two samples were uncertain. There was a difference between Gap-PCR and combined groups (Gap-PCR combined with nested PCR and MLPA) in detecting HKαα (P < 0.05). Among the 229 patients, 20 patients were identified as HKαα carriers and one was identified as -α3.7/αααanti-4.2 by two-round nested PCR and MLPA, including 15 patients with HKαα/αα, three with HKαα/αα and β-thalassemia coinheritance, one with HKαα/-SEA, one with HKαα/-α4.2 and β-thalassemia coinheritance, and one with -α3.7/αααanti-4.2 and β-thalassemia coinheritance.Conclusions:αααanti-4.2 and HKαα genotypes of patients carrying -α3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HKαα and -α3.7/αααanti-4.2 alleles. More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.

简介：AbstractBackground:With the ongoing worldwide coronavirus disease 2019 (COVID-19) pandemic, an increasing number of viral variants are being identified, which poses a challenge for nucleic acid-based diagnostic tests. Rapid tests, such as real-time reverse transcription-polymerase chain reaction (rRT-PCR), play an important role in monitoring COVID-19 infection and controlling its spread. However, the changes in the genotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARS-CoV-2 detection over time.Methods:We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2. We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern (VOCs). In addition, we also developed two rRT-PCR assays (S484K and S501Y) targeting the spike gene, which when combined with the open reading frames (ORF)1ab assay, respectively, to form duplex rRT-PCR assays, were able to detect SARS-CoV-2 VOCs (lineages B.1.351 and B.1.1.7).Results:Using a SARS-CoV-2 stock with predetermined genomic copies as a standard, the detection limit of both assays targeting RdRp and N was five copies/reaction. Furthermore, no cross-reactions with six others human CoVs (229E, OC43, NL63, HKU1, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus) were observed using these assays. In addition, the S484K and S501Y assays were combined with the ORF1ab assay, respectively.Conclusions:Four rRT-PCR assays (RdRp, N, S484K, and S501Y) were used to detect SARS-CoV-2 variants, and these assays were shown to be effective in screening for multiple virus strains.

简介：无

简介：AbstractBackground:Real-time polymerase chain reaction (PCR) is a sensitive and specific method for diagnosing schistosomiasis. However, this method should be performed in a laboratory, usually located distant from the sample collection site. Therefore, it is important to have fast sampling preservation methods, which allow simple transport prior to DNA extraction and amplification. The aim of this study was to verify if blood samples applied to filter paper are suitable for analysis of Schistosoma mansoni DNA by real-time PCR.Methods:A cross-sectional study was conducted among 100 study participants aged 17 to 70 years in a fishing village on the southern shore of Lake Victoria, Tanzania. Serum samples and ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood for preparation of dried blood spots (DBS) were collected to test for Schistosoma mansoni infection by real-time PCR. A combined diagnostic reference of positive results of serum-based real-time PCR and the Kato-Katz (KK) method was used for analysis. Sensitivity and negative predictive value (NPV) were calculated. The Wilcoxon signed-rank test was chosen to compare the mean cycle threshold (Ct) values from serum and DBS.Results:According to the reference, 92.5% S. mansoni positive samples were determined. The serum-based real-time PCR performed excellently with 95.4% sensitivity, whereas the DBS-based real-time PCR showed a low sensitivity (45.4%). The Ct-values were significantly higher in DBS (median: 37.3) than in serum samples (median: 27.5, P < 0.001), reflecting a lower parasite-specific DNA load on the filter cards. With increasing egg counts, an increase in sensitivity was observed for all methods. The POC-CCA test and the serum-based real-time PCR showed a sensitivity of 100% for medium and severe infections. The DBS real-time PCR showed a sensitivity of only 85.7% even for severe infections.Conclusions:DBS-based real-time PCR did not provide good results in our study and therefore should not be recommended or must be tested concerning temperature of storage, storage duration, use of different filter papers and extraction methods before it is used in future studies. In contrast, our results showed that the POC-CCA test is a sensitive and precise test for detecting S. mansoni infections.

简介：无

简介：Objective:Toevaluatethevalueofligasechainreaction(LCR)inthediagnosisofdiplococcusgonorrhoeaeinurine.Methods:LCRdetectionoftheurineforNeisseriagonorrhoeaeandbacteriacultureofdischargewasper-formedsimultaneouslyto276patientswithurethritisorcervicitisseekingtreatmentinsextransmitteddis-eases(STDs)outpatientclinic.Forspecimenswithdiscordantresults,polymerasechainreactionwasconducted.ThepurposewastodetecttherespectivesensitivityandspecificityofbacteriacultureandLCR.Results:24of276(8.7%)patientshadpositiveLCRresultsand21of276(7.6%)werepositiveforculture.5specimenshaddiscordantresultsfromLCRandbacteriaculture.ThesensitivityandspecificityofLCRinthediagnosisofgonorrhoeaewere92.3%and100%respectively.Conclusion:ThisstudyshowedthatLCRhadahighersensitivityandspecificityforthediagnosisofgonorrhoeaefromurine.

简介：Chainreactionbankruptcyisregardedascommonphenomenonanditseffectistobetakenintoaccountwhencreditriskportfolioisanalyzed.Butconsiderationandmodelingofitseffectleavemuchroomforimprovement.Thatismainlybecausemethodforgraspingrelationsamongcompanieswithlimiteddataisunderdeveloped.Inthisarticle,chancediscoverymethodisappliedtoestimateindustrialrelationsthataretoincludecompanies'relationsthattransmitchainreactionofbankruptcy.TimeordermethodanddirectedKeyGrapharenewlyintroducedtodistinguishandexpressthetimeorderamongdefaultsthatisessentialinformationfortheanalysisofchainreactionbankruptcy.ThestepsforthedataanalysisareintroducedandresultofexampleanalysiswithdefaultdatainKyushu,Japan,2005ispresented.Thestructureestimatedbythenewmethodiscomparedwiththestructureofactualaccountreceivableholdersofbankruptedcompaniesforevaluation.

简介：Poly(etherurethane)s(PEU),includingPEUI15andPEUH15,werepreparedthroughchain-extensionreactionofpoly(ethyleneglycol)(PEG-1500)usingdiisocyanateasachainextender,includingisophoronediisocyanate(IPDI)andhexamethylenediisocyanate(HDI).ThesePEUswereusedtotoughenpolylactide(PLA)byphysicalandreactiveblending.Thermal,morphological,mechanicalandagingpropertiesoftheblendswereinvestigatedindetail.ThesePEUswerepartiallycompatiblewithPLA.Theelongationatbreakofthereactiveblendsinthepresenceoftriphenylphosphate(TPP)forPLAwithPEUH15orPEUI15wasmuchhigherthanthatofthephysicalblends.Theagingtestwascarriedoutat-20°Cfor50hinordertoacceleratethecrystallizationofPEUs.ThePEUsinthePLA/PEUblendsproducedcrystallizationandformednewphaseseparationwithPLA,resultinginthedeclinedtoughnessofblends.Fortunately,undertheagingcondition,althoughPEUH15inblendscouldalsoformcrystallization,thereactiveblendofPLA/PEUH15/TPP(80/20/2)hadhighertoughnessthantheotherblends.TheelongationatbreakofPLA/PEUH15/TPP(80/20/2)droppedto287%fortheagingblendfrom350%fortheoriginalblend.ThetensilestrengthandmodulusofPLA/PEUH15/TPPblenddidnotchangeobviouslybecauseofthecrystallizationofPEUH15.

简介：DNA聚合酶III是为DNA的复制负责的五eubacterialDNA聚合酶之一双。在DNA聚合酶III核心酶的十个子单元之中，高山哈子单元两个都为polymerizing催化反应DNA海滨。在这研究，我们提取了高山的genomic序列哈从159的子单元定序eubacterial染色体，并且执行了基于顺序的种系发生、结构的分析。我们发现所有eubacterial染色体有至少一座高山哈子单元，哪个形式homodimers或heterodimers。种系发生并且领域高山的结构的分析以及拷贝数字变化哈在每个细菌的子单元显示高山的分类哈子单元进四个基本的组：polC，dnaE1，dnaE2，和dnaE3。这个分类具有在染色体作文分析的本质。我们也巩固了命名惯例在基因注解避免进一步的混乱。