Qiaamp dna mini kit

A total of 300 males of P. variegata obtained during Experiment A were examined for the presence of T. callipaeda L3 larvae. First, live males were taken to the laboratory, dissected under the stereo microscope (4–8×) on a Petri dish with a drop of physiological saline solution, and visually checked for the presence of the nematode in the proboscis.
Second, a total of 390 randomly chosen P. variegata males from Experiments B–D were grouped by collection date and pooled (15 individuals/pool) in vials with ethanol (70%) at −20°C for further molecular analysis. Genomic DNA was extracted from pools using a QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. Total DNA was purified using the QIAamp DNA Mini Kit (Qiagen, Germany) and stored at −20°C. The cox1 gene was partially amplified using the primer set COIintF (5’-TGATTGGTGGTTTTGGTAA-3’) and COIintR (5’-ATAAGTACGAGTATCAATATC-3’) following PCR protocol as described previously for Spirurida (Casiraghi et al. 2001 (link)). The amplified products of approximately 689 bp were analyzed by electrophoresis in 1.5% agarose gels stained with Green Safe Premium (Nzytech, Portugal), using a 100-bp DNA ladder as a molecular weight marker and observed under UV light.

Cell free circulating DNA was extracted in duplicate from 2 ml of plasma by using the QIAamp Circulating Nucleic Acid Kit (Qiagen AG, Basel, Switzerland) according to the manufacturer’s protocol. Absorbed DNA was eluted with 60 µl of provided elution buffer. The synthetic DNA RT-SPCY-T02 (Eurogentec, Angers, France) was added to the plasma to function as positive control for circulating DNA extraction. According to the manufacturer’s protocol, 2 ul of a tenfold diluted RT-SPCY-T02 was added to 2 ml of plasma. Reference buccal samples were extracted using the QIAamp DNA Mini Kit (Qiagen AG, Basel, Switzerland) according to the manufacturer’s protocol and eluted in 100 µl final volume. Both genomic and circulating DNA samples were stored at − 20 °C. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen AG Switzerland) according to the manufacturer’s guidelines and quantified using the kit QuantiFiler Trio on a QuantStudio™ 5 System (Life Technologies Europe, Zug, Switzerland). The commercial DNAs CEPH 1347-02, Control DNA 007 (Life Technologies Europe, Zug, Switzerland), 2800 M Control DNA (Promega, Dübendorf, Switzerland) were genotyped as a reference controls for allele designation.

Genomic DNA was extracted from Giemsa-stained slides, frozen blood pellets, and blood spots (summarized in Fig. 1b). Of the 119 total Group 1 samples, 117 DNA samples were from Giemsa-stained thick blood smears (stored at ambient temperature for 5 years before DNA purification) and two samples from frozen blood pellets (total volume of 100 µl) as previously reported [32 (link)]. Following extraction using the QIAamp DNA Mini Kit (Qiagen Inc., Germantown, Maryland, USA), these samples were also previously verified positive for P. falciparum using a species-specific HRM assay [32 (link)]. For Group 2, a total of 125 samples were extracted from filter paper blood spots using the QIAamp DNA Mini Kit (Qiagen Inc., Germantown, Maryland, USA). A subset of these samples were verified positive for P. falciparum as above prior to proceeding with haplotype analysis [32 (link)].

In both schools and health facilities, three capillary blood drops from finger-pricks were collected and spotted onto of Whatman 903™ filter paper (Whatman plc, UK) for PCR analysis. Whatman 903™ filter papers containing blood samples were dried and stored in individual plastic bags containing desiccant and stored at − 20 °C before transportation to Nagasaki University for PCR analysis. All DNA templates were extracted from three punched discs (6 mm in diameter) of blood spots using the QIAamp DNA Mini Kit using the QIAamp DNA Mini Kit® (Qiagen, USA) according to the manufacturer’s instructions. DNA was eluted in 50 μL of the provided buffer.

The DNA extraction method was dependent on the PCR assay and the sample matrix. For Pneumocystis-PCR, all types of respiratory samples were analysed, including bronchoalveolar lavage fluid (BALF), sputum, bronchoaspiration, nasal aspiration and pleural fluid; DNA was extracted using QIAamp DNA minikit (Qiagen) according to the manufacturer specifications. For Toxoplasma-PCR, the DNA extraction method depended on the matrix. For low cellular samples such as cerebrospinal fluids (CSF), aqueous humor, amniotic fluids and crystal clear BALF, DNA was extracted using the Tween-Nonidet-NaOH method[15 (link)]. When Pneumocystis and Toxoplasma qPCRs were performed on the same sample, QIAamp DNA minikit (Qiagen) was used. Other samples were processed using protein precipitation solution (A795A; Promega, France)[16 (link)]. As recommended, DNA extracts were then stored at– 20°C prior to PCR for best preservation[17 (link)].