Computational protocol: Comparison of Imported Plasmodium ovale curtisi and P. ovale wallikeri Infections among Patients in Spain, 2005–2011

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Protocol publication

[…] Partial sequencing of the ssrRNA gene was used to differentiate P. ovale curtisi from P. ovale wallikeri. ssrRNA amplification was performed by using a nested PCR specific for Plasmodium. The first reaction included UNR (5′-GACGGT ATCTGATCGTCTTC-3′) and PLF (5′- AGTGTGTATCCAATCGAGTTTC-3′) primers, which correspond to the first reaction of the seminested multiplex malaria PCR. The second reaction incorporated the products of the first reaction, along with NewPLFsh (5′-CTATCAGCTTTTGATGTTAG-3′) and NewRevsh (5′-CCTTAACTTTCGTTCTTG-3′) primers. Infection with different malaria species yielded products of 710–740 bp.The PCR mixture in both reactions consisted of 75 mmol/L Tris-HCl (pH 9.0), 2 mmol/L MgCl2, 50 mmol/L KCl, 20 mmol/L (NH4)2SO4, 200 μmol/L dNTP, 0.075 μmol/L of the corresponding PCR primers, 1.25 units Taq DNA polymerase (Biotools B&M Labs, S.A., Madrid, Spain), and the template DNA in a reaction volume of 50 μL. The amount of template was 5 μL of DNA extracted by using a QIAamp DNA Blood Mini Kit (QIAGEN). For the second reaction mixture, 2 μL of the PCR product of the first reaction was used as template. For both reactions, a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA) was used, beginning with 7 min at 94°C, followed by 40 cycles of 20 s at 94°C, 20 s at 62°C, and 30 s at 72°C for the first round; or 35 cycles of 20 s at 94°C, 20 s at 53°C, and 20 s at 72°C for the second round. The final cycle was followed by an extension time of 10 min at 72°C.The amplified products were purified by using Illustra DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) and sequenced by using the Big Dye Terminator v3.1 Cycle Sequencing Kit and ABI PRISM 3700 DNA Analyzer (Applied Biosystems). All amplified products were sequenced in both directions twice. To confirm P. ovale subtyping, a nested PCR amplification plus sequencing targeting cytochrome (Cyt) b was performed () in 3 samples of each group, by using a unique second amplification (nested) reaction with primers Cyt b 2F and Cyt b 2R. [...] Differences of proportions were evaluated by χ2 test or Fisher exact test, as appropriate for sample size. Means between groups were calculated by using the Student t-test for independent samples if the normal distribution could be assumed; we used the Levene test for homogeneity of variances. If normality was not valid, we used the Mann-Whitney U test for nonparametric variables.To test for normality, we used either the Shapiro-Wilks test for small samples or the Kolmogorov-Smirnov test with the Lilliefors correction for large samples. Values were reported as means and SDs or, for nonparametric distributions, medians and interquartile ranges (IQRs). A 2-sided p value of <0.05 was considered to indicate statistical significance. Statistical analyses were performed by using SPSS version 15 (SPSS Inc., Chicago, IL, USA). […]

Pipeline specifications

Software tools Biotools, SPSS
Applications Miscellaneous, Population genetic analysis
Organisms Plasmodium ovale, Homo sapiens
Diseases Infection, Thrombocytopenia