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DETECTION OF PLASMODIUM VIVAX
BY NESTED PCR AMPLIFICATION OF
DRIED BLOOD SPOTS ON FILTER PAPERS
Niu Chun
(Capital University of Medical Sciences,Beijing100054)
Zhu Xinping
(Capital University of Medical Sciences,Beijing100054)
Zhou Lei
(Capital University of Medical Sciences,Beijing100054)
Liu Qiang
(Capital University of Medical Sciences,Beijing100054)
AbstractMalaria 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.
Key wordsPlasmodium vivaxnested-PCRblood filter paperChelex
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.
1Materials and Methods
1.1Blood samples collectionA 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.
1.2DNA extraction from blood filter paper treated by Chelex-100For 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).
1.3DNA extraction from venipunctureBlood samples from venipuncture were prepared by phenol extraction and ethanol precipitation.
1.4DNA amplificationTwo 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.
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.
1.5PCR product analysis10μL 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.
2Results
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.
Fig.1The 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.
