Premix taq ex taq version 2.0 plus dye

The hypervariable V4-V5 regions of the 16S rRNA genes from bacteria and archaea were amplified in triplicate by PCR using the barcoded primers 515-F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907-R (5′-CCGTCAATTCMTTTRAGTTT-3′) (Invitrogen, Thermo Fisher Scientific Inc., Fair Lawn, USA). PCR was performed on a thermocycler using Premix Taq (EX Taq Version 2.0 plus dye, Takara Biotechnology Co. Ltd., Dalian, China) with following conditions: 94 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 10mins. The PCR products were verified by 1% agarose gel electrophoresis and quantified using GeneTools analysis software (Version 4.03.05.0, SynGene, Frederick, USA). Technical triplicate amplicons were pooled and gel purified using EZNA Gel Extraction Kit (Omega Bio-tek, Inc., Norcoross, USA). Sequencing was performed using 250-bp paired-end strategy on an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, USA) with NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, USA).

MacConkey agar medium, eosin-methylene blue agar medium, LB broth, LB agar medium, casein hydrolysate agar (MH agar, Muller-Hinton agar), and casein hydrolysate broth (MH broth, Muller-Hinton broth) were purchased from Huankai Microbial Technology, Guangzhou; DNA markers and Premix Taq (EX Taq version 2.0 plus dye) were purchased from Takara Bio, Dalian; agarose was purchased from Biowest, Spain. Antimicrobial susceptibility disks were purchased from Binhe Microorganism Reagent, Hangzhou and they were used to test susceptibility to beta-lactam antibiotics beta-lactam antibiotics (cefotaxime 30 micrograms [μg], ceftazidime 30 μg, cefoxitin 30 μg, ampicillin 10 μg, amoxicillin-clavulanate 20 μg and 10 μg), aminoglycosides (amikacin 30 μg, gentamicin 10 μg, kanamycin 30 μg, streptomycin 10 μg, neomycin 30 μg), tetracyclines (tetracycline 30 μg, doxycycline 30 μg), amphenicols (chloramphenicol 30 μg, florfenicol 30 μg), fluoroquinolones (ciprofloxacin 5 µg, enrofloxacin 5 μg, levofloxacin 5 μg), polymyxin B (300 units) and sulfisoxazole (250 μg and 300 μg).

DNA was extracted from all 40 Parascaris samples using the QIAamp DNA Mini Kit (QIAGEN, Germany) following the manufacturer’s instructions. Partial sequences of the gene of mitochondrial DNA (mtDNA), COI and ITS (including partial ITS1, 5.8 s and ITS2) were amplified to explore the genetic characteristics and phylogenetic relationships. Primer F5 (5′-TCATAAGGATATTGGGACC-3′) and primer F6 (5′-GCAAAATGTAAAGGGAAAA-3′) [17 (link)] were applied to amply the COI (996-base pair [bp]) gene of each specimen. Primers NC5 (5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and NC2 (5′-TTAGTTTCTTTTCCTCCGCT-3′) [18 (link)] were applied to amply the ITS (~770 bp) sequence of each specimen. Regarding the two PCR reactions, all the volumes were 25 μl, including 12.5 µl Premix Taq (Ex Taq version 2.0 plus dye, Takara, Japan), 8.5 µl double-distilled water ddH₂O), 1 µl of each primer and 2 µl of template DNA under the following conditions: initial denaturation at 94 °C for 5 min, then 35 cycles at 94 °C for 30 s (denaturation); annealing at 50 (COI)/55 °C (ITS) for 30 s, extension at 72 °C for 90 s (COI)/60 s (ITS), followed by a final extension at 72 °C for 7 min. The PCR product was examined on a 1.5% agarose gel to verify that the reactions produced single bands, and then was sent to Comate Biosciences Co., Ltd. (Changchun, China) for Sanger sequencing in the forward and reverse directions.