光滑念珠菌SYBR Green荧光定量PCR检测方法的建立及应用
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摘要
为建立光滑念珠菌快速准确灵敏的检测方法,根据GenBank已发表的光滑念珠菌rDNA内转录间隔区核苷酸序列设计1对特异性引物,应用SYBR Green实时荧光定量PCR方法检测光滑念珠菌,对临床分离的多株不同来源光滑念珠菌病料进行检测,通过临床常见的5种病原真菌对该方法的特异性进行检验,并用人工感染小鼠的血液、肝脏组织样本对所建方法进行检验。结果显示,该方法灵敏度高,光滑念珠菌的最低检出浓度为10 copies/μL,特异性强,与白色念珠菌、热带念珠菌、近平滑念珠菌、克柔念珠菌、烟曲霉菌等病原菌无交叉反应。该方法操作简单,耗时短,只需1 h即可完成全部检测过程。上述结果表明,SYBR Green荧光定量PCR可应用于光滑念珠菌早期侵袭的快速检测,为光滑念珠菌病的及时正确防治提供了可靠依据。
For the purpose of setting up a rapid,accurate and sensitive method to detect Candida glabrata,by means of SYBR Green fluorescent quantitative real-time PCR method,we designed a pair of special primers based on the published ITS sequences of r DNAs of Candida glabrata in Gen Bank.The clinical isolates of Candida glabrata from different sources was tested using the method.The specificity test of the method was performed using 5 clinically important pathogenic fungi.And the method was tested with blood and liver tissue samples from mice infected artificially.In result,the method was high sensitive with the lowest detection as 10 copies/μL of Candida glabrata,was high discriminative without the same reaction in the other pathogenic fungi as Candida albicans,Candida tropicalis,Candida parapsilosis,Candida krusei and Aspergillus fumigatus,respectively,and was less time consumption as long as 1 h for completing the test.In summary,this method can be used to handy and accurate detection for early infection of Candida glabrata,and it offers a reliable basis for timely and correct prevention from Candida glabrata.
For the purpose of setting up a rapid,accurate and sensitive method to detect Candida glabrata,by means of SYBR Green fluorescent quantitative real-time PCR method,we designed a pair of special primers based on the published ITS sequences of r DNAs of Candida glabrata in Gen Bank.The clinical isolates of Candida glabrata from different sources was tested using the method.The specificity test of the method was performed using 5 clinically important pathogenic fungi.And the method was tested with blood and liver tissue samples from mice infected artificially.In result,the method was high sensitive with the lowest detection as 10 copies/μL of Candida glabrata,was high discriminative without the same reaction in the other pathogenic fungi as Candida albicans,Candida tropicalis,Candida parapsilosis,Candida krusei and Aspergillus fumigatus,respectively,and was less time consumption as long as 1 h for completing the test.In summary,this method can be used to handy and accurate detection for early infection of Candida glabrata,and it offers a reliable basis for timely and correct prevention from Candida glabrata.
引文
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[17]刘建钗,马邯生,柳焕章,等.禽白念珠菌基于SYBR GreenⅠ染色的LC-PCR检测方法的建立[J].中国兽医科学,2016,46(6):799-804.LIU Jian-chai,MA Han-sheng,LIU Huan-zhang,et al.Research on SYBR GreenⅠLC-PCR technique for detecting Candida albicans in birds[J].Chinese Veterinary Science,2016,46(6):799-804.(in Chinese)
[18] SCHELL W A,BENTON J L,SMITH P B,et al.Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood[J].European Journal of Clinical Microbiology&Infectious Diseases,2012,31(9):2237-2245.
[19] NUNES C Z,MARRA A R,EDMOND M B,et al.Time to blood culture positivity as a predictor of clinical outcome in patients with Candida albicans bloodstream infection[J].BMC Infectious Diseases,2013,13:486.
[20] POILLET-PEREZ L,XIE X,ZHAN L,et al.Autophagy maintains tumour growth through circulating arginine[J].Nature,2018,DOI;10.1038/s41586-018-0697-7.
[2] MAYER F L,WILSON D,HUBE B.Candida albicans pathogenicity mechanisms[J].Virulence,2013,4(2):119-128.
[3] CORNELY O A,JOSE V,JAN D W,et al.Efficacy of micafungin in invasive candidiasis caused by common Candida species with special emphasis on non-albicans Candida species[J].Mycoses,2014,57(2):79-89.
[4] WISPLINGHOFF H,EBBERS J,GEURTZ L,et al.Nosocomial bloodstream infections due to Can dida spp.in the USA;species distribution,clinical features and antifungal susceptibilities[J].International Journal of Antimicrobial Agents,2014,43(1):78-81.
[5] BODEY G P,MARDANI M,HANNA H A,et al.The epidemiology of Candida glabrata and Candida albicans fungemia in immunocompromised patients with cancer[J].American Journal of Medicine,2002,112(5):380-385.
[6] HORN D L,NEOFYTOS DANAISSIE E J.Epidemiology and outcomes of candidemia in 2019 patients;data from the prospective antifungal therapy alliance registry[J].Clinical Infectious Diseases,2009,48(12):1695-1703.
[7] PFALLER M A,CASTANHEIRA M,LOCKHART S R,et al.Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrata[J].Journal of Clinical Microbiology,2012,50(4):1199-1203.
[8] KRCMERY V,BARNES A J.Non-albicans Candida spp.causing fungaemia;pathogenicity and antifungal resistance[J].Journal of Hospital Infection,2002,50(4):243-260.
[9]刘建钗,马邯生,柳焕章,等.肉鸡白色念珠菌的分离鉴定与基因分型[J].中国兽医科学,2015,45(10):1005-1012.LIU Jian-chai,MA Han-sheng,LIU Huan-zhang,et al.Isolation,identification and geno typing of Candida albicans from broilers chicken[J].Chinese Veterinary Science,2015,45(10):1005-1012.(in Chinese)
[10] DENNING D W,MUOZ P.Advances in invasive fungal infection and antifungal therapy;Introduction[J].Clinical Microbiology&Infection,2001,7(S2):00-00.
[11] AHMAD S,KHAN Z.Invasive candidiasis;a review of nonculture-based laboratory diagnostic methods[J].Indian Journal of Medical Microbiology,30,3(2012-08-8),2012,30(3):264.
[12] BRIZENDINE K D,BADDLEY J W,PAPPAS P G.Pulmonary cryptococcosis[J].Seminars in Respiratory&Critical Care Medicine,2011,32(06):727-734.
[13] ALEXANDER B D,JOHNSON M D,PFEIFFER C D,et al.Editor's choice;Increasing echinocandin resistance in Candida glabrata;Clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations[J].Clinical Infectious Diseases,2013,56(12):1724-1732.
[14] REDDING S W,DAHIYA M C,KIRKPATRICK W R,et al.Candida glabrata is an emerging cause of oropharyngeal candidiasis in patients receiving radiation for head and neck cancer.[J].Oral Surgery,Oral Medicine,Oral Pathology,Oral Radiology and Endodontology,2002,97(1):47-52.
[15] BAKOVL,BUCHTA V.Laboratory diagnostics of invasive fungal infections;an overview with emphasis on molecular approach[J].Folia Microbiologica,2012,57(5):421-430.
[16] PFALLER M A,SADER H S,JONES R N,et al.International surveillance of blood stream infections due to Candida species in the European SENTRY Program;species distribution and antifungal susceptibility including the investigational triazole and echinocandin agents.SENTRY Participant Group(Europe[J].Diagnostic Microbiology&Infectious Disease,1999,35(1):19.
[17]刘建钗,马邯生,柳焕章,等.禽白念珠菌基于SYBR GreenⅠ染色的LC-PCR检测方法的建立[J].中国兽医科学,2016,46(6):799-804.LIU Jian-chai,MA Han-sheng,LIU Huan-zhang,et al.Research on SYBR GreenⅠLC-PCR technique for detecting Candida albicans in birds[J].Chinese Veterinary Science,2016,46(6):799-804.(in Chinese)
[18] SCHELL W A,BENTON J L,SMITH P B,et al.Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood[J].European Journal of Clinical Microbiology&Infectious Diseases,2012,31(9):2237-2245.
[19] NUNES C Z,MARRA A R,EDMOND M B,et al.Time to blood culture positivity as a predictor of clinical outcome in patients with Candida albicans bloodstream infection[J].BMC Infectious Diseases,2013,13:486.
[20] POILLET-PEREZ L,XIE X,ZHAN L,et al.Autophagy maintains tumour growth through circulating arginine[J].Nature,2018,DOI;10.1038/s41586-018-0697-7.