Genomic dna purification kit

The sample of Onychostomalepturum was caught from the Lingshui River in Baoting County of Hainan in China (18°42'07"N, 109°40'44"E). Samples were collected from the field sites with seines, fatally anesthetized with MS-222 (Sigma, St. Louis, MO) and fixed and stored in 100% ethanol. All specimens are lodged in the laboratory of Jin-Quan Yang, Shanghai Ocean University, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources. All animal experiments were carried out in accordance with the guidelines and with approval of the Animal Research and Ethics Committee of Shanghai Ocean University (permissions, SHOU-DW-2018-021). Total genomic DNA was extracted from muscle tissue using the Genomic DNA Purification Kit (Gentra Systems, Valencia, CA) in the laboratory.

In this study, specimens were collected from eight locations in the South China region (Fig. 1). These eight locations were divided into three subregions, Zhejiang-Fujian (JO and HA), Pearl River (CX, HY and JX) and Hainan Island (QH, BS and LD) based on ichthyofaunal classification by Li [1 ]. A total of 157 specimens of G. orientalis were collected (Fig. 1; Table 1). All specimens are lodged in the laboratory of Jin-Quan Yang, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University. Specimens were collected from field sites with seines, fatally anesthetized with MS-222 (Sigma), and fixed and stored in 100 % ethanol. Genomic DNA was extracted from muscle tissue with a Genomic DNA Purification Kit (Gentra Systems, Valencia, CA).

Total DNA was extracted from whole blood, skin, uterus, ovaries, placenta, liver, spleen and umbilical cord samples using the Genomic DNA Purification Kit (Gentra Systems, Minnesota, USA), while genomic DNA from amniotic fluid and bone marrow samples was extracted using QIAampDNA Micro Kit (Qiagen, GmbH, Hilden, Germany). For each sample, two qPCR reactions were individually performed for the detection and quantification of L. infantum and A. platys nucleic acids, using primers and probes targeting, respectively, the kinetoplast minicircle DNA (kDNA) and 16S rRNA gene, as described previously [18 (link), 23 (link)]. DNA extracted from lymph nodes and whole blood from L. infantum and A. platys-infected dogs were included as positive controls. Quantification of DNA of L. infantum and A. platys was performed using a 10-fold dilution series of standard DNA from promastigotes (log phase concentration, 1.7 × 10⁶ parasites/ml) of L. infantum (zymodeme MON-1) and from A. platys-infected blood with a concentration of 5.6 × 10⁵ infected platelets/100 μl. The detection limits of the qPCRs were assessed using serial dilutions from 1.7 × 10^-2 to 1.7 × 10^-7 parasites (L. infantum) and from 2.24 × 10² to 2.24 × 10^-6 infected platelets (A. platys) per reaction (2 μl of DNA template), respectively.

The DNA of the E. anophelis isolate was prepared using a Qiagen genomic DNA purification kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany), and the genome was sequenced using an Illumina MiSeq. 2000 sequencing platform (Illumina, CA, USA). The short reads were assembled and optimized according to paired-end and overlap relationships via mapping reads to the contig using SOAP de novo. Subsystem technology prokaryotic genome annotations were based on Rapid Annotation using Subsystem Technology (RAST).

Genomic DNA was isolated from whole blood by use of Qiagen Genomic DNA Purification Kit (Qiagen Inc., Chatsworth, CA, United States). Polymerase chain reactions were carried out using HotstarTaq polymerase (Qiagen). This was done in a total volume of 20 l1 containing 1.5 mM MgCl2, 0.15 lM primers (fw: 50 -TCA CCA TCG AGA TCA ACC CC-30, rev: 50 -ACA ACG GGT CAG GCA TGC A-30), and approximately 50 ng genomic DNA. Following a 15 min denaturation step at 95°C, 45 cycles were performed including 30 s at 94°C, 30 s at 62°C, and 30 s at 72°C. PCR products were genotyped with a Pyrosequencer PSQ 96 and the PSQ 96 SNP Reagent Kit (Pyrosequencing, Uppsala, Sweden; Nordfors et al., 2002 (link)), by use of the sequence primer 50 -TGG TGG ATT TCG CTG-3.

Genomic DNA was extracted from collected tissues using Qiagen Genomic DNA Purification Kit (cat # 51304; Qiagen, Hilden, Germany) according to the manufacturer's instructions. The quality of extracted genomic DNA was measured using the NanoDrop- (ND-) 1000 Spectrophotometer V3.1.0 (NanoDrop Technologies, Inc., Wilmington, DE, USA).