Successful extraction and PCR amplification of Giardia DNA from formalin-fixed stool samples.

Extracting genomic DNA of pathogenic agents from formalin-fixed specimens is inherently difficult. Storage of samples in formalin results in nucleic acid cross-linking and DNA fragmentation. In this study, DNA was extracted from 45 Giardia-positive stool samples stored in formalin and subjected to PCR amplification targeting the triose phosphate isomerase (tpi), beta gardin (bg) and glutamate dehydrogenase (gdh) genes. Samples were rehydrated by using a descending alcohol series before DNA extraction using a commercial kit. This was followed by EDTA-mediated inhibition of DNase activity and prolonged treatment with proteinase K to digest contaminating proteins. DNA was amplified at rates of 64.4% (29/45) at the tpi, 40% (18/45) at the bg and 20% (9/45) at the gdh loci as seen on nested PCR. DNA quality was subsequently tested in a genotyping experiment which produced high-quality sequences at the tpi (41.2%; 12/29) bg (50%; 9/18), and gdh (22.2%; 2/9) loci and enabled differentiation of Giardia strains at the subtype level. The modified extraction protocol was effective at removing inhibitors and reversing cross-linking of DNA. However, PCR amplification was limited to short fragments of DNA which resulted in highest success rate on amplification of the shortest (334 bp) gene fragment tested.

A microarray platform and novel SNP calling algorithm to evaluate Plasmodium falciparum field samples of low DNA quantity.

BACKGROUND: Analysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development. In areas with adequate cold chain facilities, large volumes of leukocyte-depleted patient blood can be frozen for use in parasite genomic analyses. In more remote endemic areas smaller volumes of infected blood are taken by finger prick, and dried and stored on filter paper. These dried blood spots do not generally yield enough concentrated parasite DNA for whole-genome sequencing. RESULTS: A DNA microarray was designed for use on field samples to type a genome-wide set of SNPs which prior sequencing had shown to be variable in Africa, Southeast Asia, and Papua New Guinea. An algorithm was designed to call SNPs in samples with low parasite DNA. With this new algorithm SNP-calling accuracy of 98% was measured by hybridizing purified DNA from malaria lab strains and comparing calls with SNPs called from full genome sequences. An average accuracy of >98% was likewise obtained for DNA extracted from malaria field samples collected in studies in Southeast Asia, with an average call rate of > 82%. CONCLUSION: This new high-density microarray provided high quality SNP calls from a wide range of parasite DNA quantities, and represents a robust tool for genome-wide analysis of malaria parasites in diverse settings.

Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP).

Theileria sergenti and Theileria sinensis are closely related members of benign Theileria species found in cattle and yaks in China. They are morphologically indistinguishable. A polymerase chain reaction (PCR) targeting major piroplasm surface protein of T. sergenti and T. sinensis was developed in this study. The newly developed oligonucleotide primer set was able to specifically amplify the DNA of T. sinensis and in conjunction with primers for T. sergenti and these two species could be detected and distinguished. Specificity testing also revealed that there was no cross-reaction with the other tick-borne diseases Theileria annulata, Babesia ovata, Anaplasma marginale as well as bovine white blood cells. Phylogenetic analysis based on the MPSP gene sequences confirmed the specificity of PCR assays. The sensitivity of the methods was 0.1pg DNA for the T. sergenti PCR and 1pg DNA for T. sinensis PCR. Two hundred and thirty-six field blood samples from of cattle and yaks were collected from five different geographical regions in China where benign Theileria species have been found. T. sergenti was found in all five provinces but was absent from one county in Gansu Province. T. sinensis was only found in Gansu Province. In both counties in Gansu where the parasites co-existed, mixed infections were detected. Our results indicate that the PCR methods developed in this study are suitable for the detection and differentiation of T. sergenti and T. sinensis.

Small-scale isolation of high molecular weight DNA from Leishmania braziliensis.

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.

Diagnosis of congenital Toxoplasma gondii infection by polymerase chain reaction (PCR) on amniotic fluid samples. The Norwegian experience.

As part of a screening project for detection of Toxoplasma gondii infection among pregnant women in Norway, nested polymerase chain reaction (PCR) aimed at the detection of T. gondii in amniotic fluid samples was included in the diagnostic routine. The results were compared with the routine criteria for congenital infection: i) T. gondii detected in amniotic fluid or cord blood by mouse inoculation, ii) specific IgM or IgA in serum collected after birth, and/or iii) specific IgG persisting beyond one year of age. The PCR was based on the B1 gene with an internal control gene amplified together with the B1 gene. One hundred and two amniotic fluid samples collected during pregnancy and/or at delivery from 67 pregnant women with serological evidence of primary T. gondii infection were available for examination by both B1-PCR and mouse inoculation. Six samples were positive and 86 samples were negative by both methods (90% concordance). One sample was mouse inoculation positive and B1-PCR negative while nine samples were B1-PCR positive and mouse inoculation negative, of which five were associated with four infants without proven infection. 59%, and 41% of samples associated with infected infants were positive by B1-PCR and mouse inoculation, respectively. The difference was mainly due to a lower detection rate by mouse inoculation after antiparasitic treatment. The specificity of B1-PCR was 94%. Even though B1-PCR performed on amniotic fluid samples did not detect all infected infants, it represented a valuable tool in addition to conventional methods in the diagnosis of congenital T. gondii infection.