套式PCR检测滤纸干血滴中间日疟原虫
作者：牛春 诸欣平 周蕾 刘强
单位：牛春(首都医科大学细胞生物系寄生虫学教研室，北京 100054)；诸欣平(首都医科大学细胞生物系寄生虫学教研室，北京 100054)；周蕾(首都医科大学细胞生物系寄生虫学教研室，北京 100054)；刘强(首都医科大学细胞生物系寄生虫学教研室，北京 100054)
关键词：间日疟原虫；套式PCR；滤纸干血滴；Chelex
寄生虫与医学昆虫学报000102摘要 从滤纸干血滴上用Chelex处理洗脱下的疟原虫DNA，经套式PCR扩增间日疟原虫SSUrRNA基因特异性121 bp片段，分析该方法的敏感性和特异性。37例血样检测结果全部阳性，当原虫密度低至25个原虫/uL血时仍可成功检测到该特异条带，且其它三种人疟原虫(恶性疟原虫，三日疟原虫和卵形疟原虫)血样均为阴性。提示滤纸干血滴与PCR扩增技术相结合，是疟疾诊断或流行病学调查的实用工具。
, http://www.100md.com
DETECTION OF PLASMODIUM VIVAX
BY NESTED PCR AMPLIFICATION OF
DRIED BLOOD SPOTS ON FILTER PAPERS
Niu Chun
(Capital University of Medical Sciences,Beijing 100054)
Zhu Xinping
(Capital University of Medical Sciences,Beijing 100054)
Zhou Lei
, 百拇医药
(Capital University of Medical Sciences,Beijing 100054)
Liu Qiang
(Capital University of Medical Sciences,Beijing 100054)
Abstract Malaria parasites DNA was eluted from dried blood spot on filter paper by Chelex treatment and amplified by PCR to detect low parasitemia of Plasmodium vivax. Sensitivity and specificity of nested PCR assay were confirmed by amplifying a 121 bp DNA fragment of SSUrRNA gene of Plvivax.Density of about 25 parasites per ul of blood can be detected successfully.None of other three human malaria species(Plasmodium falciparum,Plasmodium malariae and Plasmodium ovale)samples was positive.The ease of collection and transport of filter paper specimens combined with the sensitive and specific detection of P.vivax by nested PCR suggest that this method might be a valuable tool for moleculr epidemiological study of P.vivax.
, 百拇医药
Key words Plasmodium vivax nested-PCR blood filter paper Chelex
Polymerized chain reaction(PCR)assay is sensitive and specific in malaria diagnosis.The main limitations of PCR for the detection of Plasmodium infections have been the requirement for labor-intensive organic extraction of DNA from whole blood and the difficulties in collecting and transporting suitable samples from the field (Jaureguiberry,et al.,1990 and Kimura,et al.,1990).Here we used a simple and quick method to solve these problems by collecting finger-prick samples in the field and dotting directly onto filter paper.Parasites DNA can be eluted from these dry blood dots and amplified by PCR to detect of P.vivax.A nested PCR procedure which amplified a 121 bp DNA of a SSUrRNA gene specific to Plasmodium viviax was sued to identify the sensitivity and specificity of this method.The results demonstrated that the simple dried blood spot sampling,when coupled with nested PCR is more convenient and suitable for detecting malaria in large scale of epidemiological surveys.
, 百拇医药
1 Materials and Methods
1.1 Blood samples collection A total of 37 finger-prick blood samples was obtained from patients in Minshan,Sichuan province,one of the malaria endemic regions in China.Two set of thick and thin blood films were prepared from each individual together with duplicate finger-prick blood samples,each between 20～30μL,which were spotted directly onto filter paper(Whatman 3 MM chromatography paper,2 cm×6 cm).Filter paper samples were air dried,individually placed in plastic bags and mailed at room temperature to Capital University of Medical Sciences where they were stored at room temperature until processed.Three control blood samples were obtained from venipuncture.P.malariae blood sample and P.ovale blood sample were obtained from Minshan,Sichuan province two years ago and over 10 years respectively,and stored at -80℃ until processed.P.falciparum blood sample were obtained from Hainan province and stored as above until processed.
, 百拇医药
1.2 DNA extraction from blood filter paper treated by Chelex-100 For each sample to be processed,180uL of a 5%(wt/vol)Chelex-100 solution(Bio-Rad.Catalog no.1422832)was added to a 1.5 mL microcentrifuge tube and placed in a heating block at 100℃ for 10 min.Each filter paper sample(approximately 1 cm in diameter)was excised with a new,clean razor blade and added to the hot Chelex-solution.The tube was capped,gently,vortexed for 30s,and returned to the heat block for 1 min.The samples were centrfuged (12 000g for 2 min),and the supernatant was removed to a new microcentrifuge tube and spun again(12 000 g for 2 min).Finally,the supernatant was removed to a new microcentrifuge tube and either used immediately in an amplification reaction or stored at 4℃ until required(Kevin,et al.,1991).
, 百拇医药
1.3 DNA extraction from venipuncture Blood samples from venipuncture were prepared by phenol extraction and ethanol precipitation.
1.4 DNA amplification Two primers pairs were used in nested PCR reaction.In the first reaction,the primers used were rPLU5(5'-CCTGTTGTTGCCTTAAACTTC-3')and rPLU6(5'-TTAAAATTGTTGCAGTTAAAACG-3'),which amplified Plasmodium genus-specific SSUrRNA gene,and in the second reaction,rVIV1(5'-CGCTTCTAGCTT-AATCCACATAACTGATAC-3')and rVIV2(5'-ACTTCCAAGCCGAAGCAA-AGAAAGTCCTTA-3')were used,which amplified Plasmodium vivax species-specific SSUrRNA gene,all these primers were described by Snounou et al.(1993)and synthesized by Cydersyn (Beijing) Co. Ltd.
, 百拇医药
DNA prepared as described above was amplified in 20μL of reaction mixture containing 2μL DNA template,2.5 mM MgCl2,125μM dNTP,2.5μM each of primers,and 0.8 unit Tag polymerase.The reaction mixture was over laid with 15μL mineral oil.The amplification program was as follow:Step 1,95℃ for 5 min;Step 2,annealing at 58℃ for 2 min;Step 3,extension at 72℃ for 2 min;Step 4,denaturation at 94℃ for 1min;repeat Steps 2～4 29 times,then Step 2,and finally Step 3 for 5 min. 2μL of the product obtained was then used as a template in a second amplification reaction.
, 百拇医药
1.5 PCR product analysis 10μL reaction product was detected by electrophoresis on 2% agarose gels.Agarose gels were made in TBE buffer and run under 65 voltage for about 2 hours.Target DNA was visualized on an Gel documentation system(UVP GS 8000)following ethidium bomide staining.
2 Results
Sensitivity of the assay DNA templates extracted from blood filter paper treated by Chelex-100 contained nearly 2 500 parasites per μL were ten-fold servial diluted with sterile water to a final number of parasites ranging from 0.25,to 250 parasites per μL.Then 2μL extraction were used as templates in all reaction.The results showed that template containing as few as 25 parasites per μL showed clearly visible 121 bp band. Fig 1.
, http://www.100md.com
Fig.1 The detection limit of Plasmodium vivas by dried blood filter paper
Lane 1:100bp DNA ladder. Lane 2～6:respectivel
y 2 500,250,25,2.5，0.25 parasites per uL of template.
Specificity of the assay:Control genomic DNAs from Plasmodium falciparum (blood filter),Plasmodium ovale and Plasmodium malariae(Venipuncture)were used as control to confirm the specificity of this assay.As is shown in Fig 2.,electrophoresis indicated that the 121 bp DNA fragment specific to Plasmodium vivax SSUrRNA gene only be detected in sample of Plasmodium vivax,and no such fragment is visible with the P.falciparum,P.ovale and P.malariae DNA templates.
, 百拇医药
Fig.2 The specificity of Plasmodium vivas by nested PCR of dried blood filter paper
Lane 1:100bp DNA ladder. Lane 2:negative control. Lane 3:Plasmodium vivax. Lane
4:Plasmodium malariae. Lane 5:Plasmodium falciparum. Lane 6:Plasmodium ovate.
Positive ratio in clinical patients All of the 37 blood filter paper samples from patients treated by Chelex-100 showed the target band.The positive concordance rate between nested PCR and microscopy was 100% in detecting P.vivax from field.
, 百拇医药
3 Discussion
Microscopy is of present the method of choice for detecting P.vivax.However,even for an expert microscopist,this method is time consuming and labor intensive,and it is difficult to identify mixed P.falciparum and P.vivax infections if only ring stages are present.Alternative approaches for the detection and characterization of P.falciparum have included in vitro culture and DNA probes;but neither of these methods has been as successful for the study of P.vivax.Reliable in vitro cultivation of P.vivax has not been achieved and DNA probe used in direct assays for the detection of P.vivax infections have been at least 10-fold less sensitive than that for P.falciprum(Roy,et al.,1987,Barker,et al.,1990 and 1992).PCR has greatly facilitated the diagnosis of plasmodia.However,there are still some problems:the presence of iron and other metals may inhibit enzymatic amplification of DNA.Techniques to avoid this inhibition often rely upon tedious procedures,including erythrocytelysis,proteinase K digestion,phenol-chloroform extractions,ethanol precipitation.In addition,they require either fresh whole-blood samples or freshly frozen and transported venipuncture blood samples,making the collection and transportation of samples from field sites difficult.Foreign scientists demonstrated that P.falciparum DNA can be efficiently extracted and amplified from blood-dotted filter paper.They used Chelex-100 in the extraction process to bind ions that may be inhibitory to Taq DNA polymerase(Kevin et al.,1993; Philpott et al.,1990).This approach avoids many difficulties associated with collecing,transporting,and processing specimens from field location.Wan et al.(1995) and Sun et al. (1996) simplified this process.They use sterile water-boiling instead of Chelex-boiling in detecting P.falciparum infection.Although the detection limitation of his approach is 0.5 parasite per uL blood,PCR was occasionally negative in patients who were smear-positive.In addition,we found in this study that the stability of sterile water-boiling approach rely deeply on the hours of soak.When the blood spotted filter papers were soaked less than 6 hours,the positive ratio in clinical patients decreased to 10%～20%.it suggested that sterile water-boiling may not remove the inhibitors as good as Chelex does.As we know,P.vivax generally exhibits lower parasitemias than does P.falciparum (Barker,et al.,1990).So we choose Chelex to treat blood filter paper as described by Kevin et al. and improved it as follows:the volume of template was only 2 μL(corresponds to about 0.2 μL whole blood),and the volume of reaction mixture was decreased to 20 μL to save on expensive Taq DNA polymerase.With 30 cycles of amplification,we were able to detect 25 parasites per μL blood.None of three control blood samples were positive.For the more,the time used in preparing DNA template with tiddley Chelex(cost about $0.01 per sample)were only 20 min.It means that this approach offers the advantages of ease of preparation of template,reliability of results and economy of reagent and sample volume which is very suitable to the state of China.
, http://www.100md.com
In summary,the ease of collection and transport of filter paper speciments combined with the sensitive and specific detection of P.vivax by nested PCR suggest that this method might be a valuable tool for epidemiological study of P.vivax.
国家教委留学回国人员科研启动基金资助项目、北京市教委科技发展基金资助项目。
References
1，Kevin,C.K.and L.DavidE.1991 Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood.J.Clin.Micro.June:1171～1174.
, http://www.100md.com
2，Snounou,G.,S.Viryakosol,X.P.Zhu et al.1993 High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction.Mol.Biochem.Parasitol.61(2):315～320.
3，Roy,K.B.,V.Yajnik,A.Roy et al.1987 Detection of P.vivax in human blood using synthetic DNA probe.Indian J.Malar. 24:63～69.
4，Barker,R.H.Jr.1990 DNA probe diagnosis of parasitic infections.Exp.Parasitol.70: 494～499.
, 百拇医药
5，Barker,R.H.Jr.,T.Banchongaksorn,J.M.Courval et.al. 1992 A simple method to detect Plasmodium falciparum directly from blood samples using the polymerase chain reaction.Am.J.Trop.Med.Hyg.46:416～426.
6，Kevin,C.K.,E.B.Arthur,M.Laura et al.1993 Detection of Plasmodium vivax by polymerase chain reaction in a field study.J.Inf.Dis.168:1323～1326.
7，Philpott,J.,J.S.Keystone,A.Reid et al.1990 Effect of malaria chemoprophylaxis on the development of antibodies to Plasmodium falciparum in expatriates living in West Africa.Am.J.Trop.Med.Hyg.42:28～35.
, 百拇医药
8，Wan,L.,P.X.Chen,C.F.Xue et al.1995 Studies on diagnosis of falciparum malaria based on amplifiying specific SSUrDNA fragment with nested PCR.Chin.J.Parasitol.& Parasitic Dis.13(3):174～177.
9，Sun,M.L.,P.X.Chen,C.F.Xue et al.1996 Detection of Plasmodium falciparum from dried-blood-spotted filter paper by nested PCR.Chin.J.Parasitol.& Parasitic Dis.14(3):197～200.
收稿日期：1999-02-10, 百拇医药
单位：牛春(首都医科大学细胞生物系寄生虫学教研室，北京 100054)；诸欣平(首都医科大学细胞生物系寄生虫学教研室，北京 100054)；周蕾(首都医科大学细胞生物系寄生虫学教研室，北京 100054)；刘强(首都医科大学细胞生物系寄生虫学教研室，北京 100054)
关键词：间日疟原虫；套式PCR；滤纸干血滴；Chelex
寄生虫与医学昆虫学报000102摘要 从滤纸干血滴上用Chelex处理洗脱下的疟原虫DNA，经套式PCR扩增间日疟原虫SSUrRNA基因特异性121 bp片段，分析该方法的敏感性和特异性。37例血样检测结果全部阳性，当原虫密度低至25个原虫/uL血时仍可成功检测到该特异条带，且其它三种人疟原虫(恶性疟原虫，三日疟原虫和卵形疟原虫)血样均为阴性。提示滤纸干血滴与PCR扩增技术相结合，是疟疾诊断或流行病学调查的实用工具。
, http://www.100md.com
DETECTION OF PLASMODIUM VIVAX
BY NESTED PCR AMPLIFICATION OF
DRIED BLOOD SPOTS ON FILTER PAPERS
Niu Chun
(Capital University of Medical Sciences,Beijing 100054)
Zhu Xinping
(Capital University of Medical Sciences,Beijing 100054)
Zhou Lei
, 百拇医药
(Capital University of Medical Sciences,Beijing 100054)
Liu Qiang
(Capital University of Medical Sciences,Beijing 100054)
Abstract Malaria parasites DNA was eluted from dried blood spot on filter paper by Chelex treatment and amplified by PCR to detect low parasitemia of Plasmodium vivax. Sensitivity and specificity of nested PCR assay were confirmed by amplifying a 121 bp DNA fragment of SSUrRNA gene of Plvivax.Density of about 25 parasites per ul of blood can be detected successfully.None of other three human malaria species(Plasmodium falciparum,Plasmodium malariae and Plasmodium ovale)samples was positive.The ease of collection and transport of filter paper specimens combined with the sensitive and specific detection of P.vivax by nested PCR suggest that this method might be a valuable tool for moleculr epidemiological study of P.vivax.
, 百拇医药
Key words Plasmodium vivax nested-PCR blood filter paper Chelex
Polymerized chain reaction(PCR)assay is sensitive and specific in malaria diagnosis.The main limitations of PCR for the detection of Plasmodium infections have been the requirement for labor-intensive organic extraction of DNA from whole blood and the difficulties in collecting and transporting suitable samples from the field (Jaureguiberry,et al.,1990 and Kimura,et al.,1990).Here we used a simple and quick method to solve these problems by collecting finger-prick samples in the field and dotting directly onto filter paper.Parasites DNA can be eluted from these dry blood dots and amplified by PCR to detect of P.vivax.A nested PCR procedure which amplified a 121 bp DNA of a SSUrRNA gene specific to Plasmodium viviax was sued to identify the sensitivity and specificity of this method.The results demonstrated that the simple dried blood spot sampling,when coupled with nested PCR is more convenient and suitable for detecting malaria in large scale of epidemiological surveys.
, 百拇医药
1 Materials and Methods
1.1 Blood samples collection A total of 37 finger-prick blood samples was obtained from patients in Minshan,Sichuan province,one of the malaria endemic regions in China.Two set of thick and thin blood films were prepared from each individual together with duplicate finger-prick blood samples,each between 20～30μL,which were spotted directly onto filter paper(Whatman 3 MM chromatography paper,2 cm×6 cm).Filter paper samples were air dried,individually placed in plastic bags and mailed at room temperature to Capital University of Medical Sciences where they were stored at room temperature until processed.Three control blood samples were obtained from venipuncture.P.malariae blood sample and P.ovale blood sample were obtained from Minshan,Sichuan province two years ago and over 10 years respectively,and stored at -80℃ until processed.P.falciparum blood sample were obtained from Hainan province and stored as above until processed.
, 百拇医药
1.2 DNA extraction from blood filter paper treated by Chelex-100 For each sample to be processed,180uL of a 5%(wt/vol)Chelex-100 solution(Bio-Rad.Catalog no.1422832)was added to a 1.5 mL microcentrifuge tube and placed in a heating block at 100℃ for 10 min.Each filter paper sample(approximately 1 cm in diameter)was excised with a new,clean razor blade and added to the hot Chelex-solution.The tube was capped,gently,vortexed for 30s,and returned to the heat block for 1 min.The samples were centrfuged (12 000g for 2 min),and the supernatant was removed to a new microcentrifuge tube and spun again(12 000 g for 2 min).Finally,the supernatant was removed to a new microcentrifuge tube and either used immediately in an amplification reaction or stored at 4℃ until required(Kevin,et al.,1991).
, 百拇医药
1.3 DNA extraction from venipuncture Blood samples from venipuncture were prepared by phenol extraction and ethanol precipitation.
1.4 DNA amplification Two primers pairs were used in nested PCR reaction.In the first reaction,the primers used were rPLU5(5'-CCTGTTGTTGCCTTAAACTTC-3')and rPLU6(5'-TTAAAATTGTTGCAGTTAAAACG-3'),which amplified Plasmodium genus-specific SSUrRNA gene,and in the second reaction,rVIV1(5'-CGCTTCTAGCTT-AATCCACATAACTGATAC-3')and rVIV2(5'-ACTTCCAAGCCGAAGCAA-AGAAAGTCCTTA-3')were used,which amplified Plasmodium vivax species-specific SSUrRNA gene,all these primers were described by Snounou et al.(1993)and synthesized by Cydersyn (Beijing) Co. Ltd.
, 百拇医药
DNA prepared as described above was amplified in 20μL of reaction mixture containing 2μL DNA template,2.5 mM MgCl2,125μM dNTP,2.5μM each of primers,and 0.8 unit Tag polymerase.The reaction mixture was over laid with 15μL mineral oil.The amplification program was as follow:Step 1,95℃ for 5 min;Step 2,annealing at 58℃ for 2 min;Step 3,extension at 72℃ for 2 min;Step 4,denaturation at 94℃ for 1min;repeat Steps 2～4 29 times,then Step 2,and finally Step 3 for 5 min. 2μL of the product obtained was then used as a template in a second amplification reaction.
, 百拇医药
1.5 PCR product analysis 10μL reaction product was detected by electrophoresis on 2% agarose gels.Agarose gels were made in TBE buffer and run under 65 voltage for about 2 hours.Target DNA was visualized on an Gel documentation system(UVP GS 8000)following ethidium bomide staining.
2 Results
Sensitivity of the assay DNA templates extracted from blood filter paper treated by Chelex-100 contained nearly 2 500 parasites per μL were ten-fold servial diluted with sterile water to a final number of parasites ranging from 0.25,to 250 parasites per μL.Then 2μL extraction were used as templates in all reaction.The results showed that template containing as few as 25 parasites per μL showed clearly visible 121 bp band. Fig 1.
, http://www.100md.com
Fig.1 The detection limit of Plasmodium vivas by dried blood filter paper
Lane 1:100bp DNA ladder. Lane 2～6:respectivel
y 2 500,250,25,2.5，0.25 parasites per uL of template.
Specificity of the assay:Control genomic DNAs from Plasmodium falciparum (blood filter),Plasmodium ovale and Plasmodium malariae(Venipuncture)were used as control to confirm the specificity of this assay.As is shown in Fig 2.,electrophoresis indicated that the 121 bp DNA fragment specific to Plasmodium vivax SSUrRNA gene only be detected in sample of Plasmodium vivax,and no such fragment is visible with the P.falciparum,P.ovale and P.malariae DNA templates.
, 百拇医药
Fig.2 The specificity of Plasmodium vivas by nested PCR of dried blood filter paper
Lane 1:100bp DNA ladder. Lane 2:negative control. Lane 3:Plasmodium vivax. Lane
4:Plasmodium malariae. Lane 5:Plasmodium falciparum. Lane 6:Plasmodium ovate.
Positive ratio in clinical patients All of the 37 blood filter paper samples from patients treated by Chelex-100 showed the target band.The positive concordance rate between nested PCR and microscopy was 100% in detecting P.vivax from field.
, 百拇医药
3 Discussion
Microscopy is of present the method of choice for detecting P.vivax.However,even for an expert microscopist,this method is time consuming and labor intensive,and it is difficult to identify mixed P.falciparum and P.vivax infections if only ring stages are present.Alternative approaches for the detection and characterization of P.falciparum have included in vitro culture and DNA probes;but neither of these methods has been as successful for the study of P.vivax.Reliable in vitro cultivation of P.vivax has not been achieved and DNA probe used in direct assays for the detection of P.vivax infections have been at least 10-fold less sensitive than that for P.falciprum(Roy,et al.,1987,Barker,et al.,1990 and 1992).PCR has greatly facilitated the diagnosis of plasmodia.However,there are still some problems:the presence of iron and other metals may inhibit enzymatic amplification of DNA.Techniques to avoid this inhibition often rely upon tedious procedures,including erythrocytelysis,proteinase K digestion,phenol-chloroform extractions,ethanol precipitation.In addition,they require either fresh whole-blood samples or freshly frozen and transported venipuncture blood samples,making the collection and transportation of samples from field sites difficult.Foreign scientists demonstrated that P.falciparum DNA can be efficiently extracted and amplified from blood-dotted filter paper.They used Chelex-100 in the extraction process to bind ions that may be inhibitory to Taq DNA polymerase(Kevin et al.,1993; Philpott et al.,1990).This approach avoids many difficulties associated with collecing,transporting,and processing specimens from field location.Wan et al.(1995) and Sun et al. (1996) simplified this process.They use sterile water-boiling instead of Chelex-boiling in detecting P.falciparum infection.Although the detection limitation of his approach is 0.5 parasite per uL blood,PCR was occasionally negative in patients who were smear-positive.In addition,we found in this study that the stability of sterile water-boiling approach rely deeply on the hours of soak.When the blood spotted filter papers were soaked less than 6 hours,the positive ratio in clinical patients decreased to 10%～20%.it suggested that sterile water-boiling may not remove the inhibitors as good as Chelex does.As we know,P.vivax generally exhibits lower parasitemias than does P.falciparum (Barker,et al.,1990).So we choose Chelex to treat blood filter paper as described by Kevin et al. and improved it as follows:the volume of template was only 2 μL(corresponds to about 0.2 μL whole blood),and the volume of reaction mixture was decreased to 20 μL to save on expensive Taq DNA polymerase.With 30 cycles of amplification,we were able to detect 25 parasites per μL blood.None of three control blood samples were positive.For the more,the time used in preparing DNA template with tiddley Chelex(cost about $0.01 per sample)were only 20 min.It means that this approach offers the advantages of ease of preparation of template,reliability of results and economy of reagent and sample volume which is very suitable to the state of China.
, http://www.100md.com
In summary,the ease of collection and transport of filter paper speciments combined with the sensitive and specific detection of P.vivax by nested PCR suggest that this method might be a valuable tool for epidemiological study of P.vivax.
国家教委留学回国人员科研启动基金资助项目、北京市教委科技发展基金资助项目。
References
1，Kevin,C.K.and L.DavidE.1991 Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood.J.Clin.Micro.June:1171～1174.
, http://www.100md.com
2，Snounou,G.,S.Viryakosol,X.P.Zhu et al.1993 High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction.Mol.Biochem.Parasitol.61(2):315～320.
3，Roy,K.B.,V.Yajnik,A.Roy et al.1987 Detection of P.vivax in human blood using synthetic DNA probe.Indian J.Malar. 24:63～69.
4，Barker,R.H.Jr.1990 DNA probe diagnosis of parasitic infections.Exp.Parasitol.70: 494～499.
, 百拇医药
5，Barker,R.H.Jr.,T.Banchongaksorn,J.M.Courval et.al. 1992 A simple method to detect Plasmodium falciparum directly from blood samples using the polymerase chain reaction.Am.J.Trop.Med.Hyg.46:416～426.
6，Kevin,C.K.,E.B.Arthur,M.Laura et al.1993 Detection of Plasmodium vivax by polymerase chain reaction in a field study.J.Inf.Dis.168:1323～1326.
7，Philpott,J.,J.S.Keystone,A.Reid et al.1990 Effect of malaria chemoprophylaxis on the development of antibodies to Plasmodium falciparum in expatriates living in West Africa.Am.J.Trop.Med.Hyg.42:28～35.
, 百拇医药
8，Wan,L.,P.X.Chen,C.F.Xue et al.1995 Studies on diagnosis of falciparum malaria based on amplifiying specific SSUrDNA fragment with nested PCR.Chin.J.Parasitol.& Parasitic Dis.13(3):174～177.
9，Sun,M.L.,P.X.Chen,C.F.Xue et al.1996 Detection of Plasmodium falciparum from dried-blood-spotted filter paper by nested PCR.Chin.J.Parasitol.& Parasitic Dis.14(3):197～200.
收稿日期：1999-02-10, 百拇医药