title: Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP Products As Determined by Liquid Chromatography/Quadrupole-Orbitrap Hybrid Mass Spectrometry.
abstract: A method based on liquid chromatography-electrospray mass spectrometric analysis of the enzymatically digested amplicons derived from the mitochondrial 16S rRNA gene was established for the discrimination of toxic pufferfish. A MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q Exactive mass spectrometer (Thermo Fisher) were employed for separation and detection, respectively. Monoisotopic masses of the oligonucleotides were calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm on average) and precision (relative standard deviation, 0.49 ppm on average) were achieved, and a mass accuracy of <2.8 ppm was maintained for at least 180 h without additional calibration. The present method was applied to 29 pufferfish samples, and results were consistent with Sanger sequencing.
authors.affiliation: National Research Institute of Police Science , 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Miyaguchi
authors.affiliation: National Research Institute of Police Science , 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yamamuro
authors.affiliation: National Research Institute of Police Science , 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors.forename: Hikoto
authors.initials: H
authors.lastname: Ohta
authors.affiliation: National Research Institute of Police Science , 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakahara
authors.affiliation: National Research Institute of Police Science , 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005838:Genotype; D013058:Mass Spectrometry / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D023721:Tetraodontiformes / Q000145:classification / Q000235:genetics*
chemical_list: D012336:RNA, Ribosomal, 16S
keywords: electrospray ionization mass spectrometry; fugu; polymerase chain reaction; pufferfish; restriction fragment length polymorphism