内脏利什曼病患者骨髓及血内病原体特异性kDNA片段PCR扩增用于诊断的研究

目的 :建立简易、准确的诊断内脏利什曼病和病原体鉴定技术。方法 :采用作者设计的杜氏利什曼原虫 ( L.d.)种特异引物 和 ,经 PCR扩增样品内病原体 k DNA2 97bp片段 ,检测 2 2例确诊内脏利什曼病 ( VL )患者骨髓、血、血清共 55份样品和 4例临床疑诊为黑热病患者的骨髓 (共 2 6例 ,59份样品 )。结果 :( 1) PCR法与骨髓涂片镜检符合率为 96.2 % ( 2 5/2 6) ;( 2 ) PCR法检测骨髓、血及血清的总阳性率为 95.4 % ( 2 1/2 2 ) ;( 3) PCR法检测 2 2例骨髓、 16例血样品及 17例血清样品 ,阳性率分别为 91% ( 2 0 /2 2 )、 68.8% ( 11/16)及 2 9.4 % ( 5/17)。 15例骨髓对照样品得自 9例白血病患者及 6例健康人。血及血清对照样品各得自 5例健康人。全部对照未见扩增产物 ,均为阴性。结论 :采用引物 和 进行 PCR扩增检测血内 k DNA特异片段诊断内脏利什曼病有较好前景。

STUDIESON DIAGNOSISOF VISCERAL LEISHMANIASIS BASED ON PCR AMPLIFICATION OF LEISHMANIA DONOVANI k DNA FRAGMENT FROM BONE MARROW AND BLOOD SAMPLES OF VISCERAL L EISHMANIASISPATIENTS

AIM:To develop a simple and accurate technique for the diagnosis of visceral leishmani- asis (VL ) and identification of Leishmania pathogen. METHODS:Based on a set of oligonu- cleotide primers I and II designed by the authors with L.donovani specificity,a polymerase chain reaction(PCR) was conducted to amplify a minicircle k DNA fragment (2 97bp) for i- dentification and detection of L.donovani in the bone marrow,blood and serum samples from 2 2 confirmed VL patients and4bone marrow samples from4clinically diagnosed cases(5 9 samples collected from2 6 cases) .RESUL TS:(1 ) The coincidence rate between PCR and bone marrow smear method was96 .2 % (2 5 / 2 6 ) ;(2 ) The total positivity of PCR with the bone marrow,blood and serum was 95 .4% (2 1 / 2 2 ) ;(3) The positive rate of PCR amplifica- tion with bone marrow,blood and serum specimens were91 .0 % (2 0 / 2 2 ) ,6 8.8% (1 1 / 1 6 ) and 2 9.4% (5 / 1 7) ,respectively. The controls included 1 5 bone marrow samples from 9 leukemia patients and6 normal persons;5 blood and5 serum samples of normal persons.No amplification productwas showed from all of the controls.CONCLUSION:The resultshows that the diagnosis of visceral leishmaniasis based on detecting k DNA in blood by PCR ampli- fication with primers I and II is promising.