Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP

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Genotyping of toxic pufferfish based on specific PCRRFLP products as determined by liquid chromatography/ quadrupole-Orbitrap hybrid mass spectrometry Hajime Miyaguchi, Tadashi Yamamuro, Hikoto Ohta, Hiroaki Nakahara, and Shinichi Suzuki J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03703 • Publication Date (Web): 02 Oct 2015 Downloaded from http://pubs.acs.org on October 2, 2015

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Journal of Agricultural and Food Chemistry

Research Article Genotyping of toxic pufferfish based on specific PCR-RFLP products as determined by liquid chromatography/quadrupole-Orbitrap hybrid mass spectrometry Hajime Miyaguchi*, Tadashi Yamamuro, Hikoto Ohta, Hiroaki Nakahara and Shinichi Suzuki National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan *Corresponding Author. Tel.: +81471358001; Fax: +81471339189; E-mail: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT: A method for the discrimination of toxic pufferfish was established

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based on liquid chromatography/electrospray mass spectrometric analysis of the

3

enzymatically digested amplicons derived from mitochondrial 16S rRNA gene. A

4

MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q

5

Exactive mass spectrometer (Thermo Fisher) were employed for separation and

6

detection, respectively. Monoisotopic masses of the oligonucleotides were

7

calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although

8

a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm

9

on average) and precision (relative standard deviation, 0.49 ppm on average)

10

were achieved and a mass accuracy of less than 2.8 ppm was maintained for at

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least 180 hours without additional calibration. The present method was applied

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to 29 pufferfish samples and results were consistent with Sanger sequencing.

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KEYWORDS: Pufferfish, Fugu, Polymerase chain reaction, Restriction fragment

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length polymorphism, Electrospray ionization mass spectrometry

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INTRODUCTION

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According to an official bulletin by the Japanese government, 21 species of

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pufferfish are categorized as edible; however, not all parts (organs/tissues) are

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edible. The edible parts that are free of tetrodotoxin vary by species. Other

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pufferfish are prohibited for sale because of the presence of toxins throughout

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the whole body (e.g., Lagocephalus lunaris1) or because there is a lack of

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toxicity data for a particular species. Therefore, species-based labeling of

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pufferfish is officially required in the Japanese market; mislabeling, which could

25

lead to serious food poisoning, is a criminal offence.

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Differentiation and discrimination of pufferfish is not just a challenge in Japan. In

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the United States, the distribution of pufferfish is prohibited except for limited

28

importation from Japan of edible parts of Takifugu rubripes. However, a

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tetrodotoxin poisoning occurred in Chicago in 2007 because of an illegal

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importation of frozen pufferfish (Lagocephalus family) from China that had been

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mislabeled as monkfish.2

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Discrimination of pufferfish species is carried out in the Japanese market

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morphologically, however, inedible L. lunaris is at risk of misidentification as

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edible L. wheeleri, owing to the species having similar morphologies.1 Moreover,

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morphological examination of pufferfish is virtually impossible in the case of

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filleted or minced fish. A tetrodotoxin poisoning outbreak due to consumption of

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dried fillets of L. lunaris occurred in Minneapolis in 2014.3 A discrimination

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capability is also needed in the context of food fraud investigations because

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Takifugu rubripes is far more expensive than other kinds of pufferfish. 3



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For DNA-based differentiation of pufferfish species, the genes of cytochrome b,

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12S rRNA, 16S rRNA and cytochrome c oxidase I in mitochondrial DNA have

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been analyzed by Sanger sequencing, PCR-restriction fragment length

43

polymorphism (RFLP) analysis and real-time PCR analysis.2,4–8 While Sanger

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sequencing is accurate and well-established, PCR-RFLP and real-time PCR

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analysis are simple and rapid for analyzing DNA polymorphism. However,

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because detection of DNA in the latter two methods is usually carried out by

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non-sequence-based techniques which typically involve using a DNA-binding

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fluorescent dye, the reliability of discrimination is inferior to that of Sanger

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sequencing.

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Mass spectrometry (MS) is one of the most reliable technologies for identifying

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and characterizing biomolecules. Although matrix-assisted laser

52

desorption/ionization–time-of-flight MS is frequently used for the analysis of

53

proteins and short oligonucleotides (less than ~25 nucleotides (nt)), it is not

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applicable to long oligonucleotides owing to the subsequent fragmentation,

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adduct formation and low ionization efficiency.9–11 Instead, electrospray

56

ionization (ESI) can produce many charge states of multiply charged ions

57

([M-nH]n-) from biomacromolecules and, therefore, the mass of the analyte can

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exceed the upper limit of the scan range of the spectrometer. This requires

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transformation of a charge state series into the corresponding mass via

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deconvolution. In the context of ESI-MS, whereas dissociation of a DNA duplex,

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purification and desalting are required for direct measurement to overcome

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ionization issues such as ion suppression and adduct formation,12–17 liquid 4


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chromatography (LC) provides an on-line separation of the analytes from

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coexisting substances instead of laborious sample purification and desalting.18,19

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Whereas LC/ESI-MS has been used for amplicon-based genotyping including

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differentiation of pathogen species, single nucleotide polymorphism and short

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tandem repeat,9,12,15,20–24 discrimination of species other than pathogens by

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LC/ESI-MS has not been reported.

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In this study, LC/ESI-MS is applied for the first time for PCR-RFLP analysis of

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fish samples to permit rapid and accurate discrimination of toxic pufferfish

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species.

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MATERIALS AND METHODS

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Chemicals and materials

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1,1,1,3,3,3-Hexafluoro-2-propanol and triethylamine were “eluent additive for

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LC-MS” grade and supplied by Sigma–Aldrich (St. Louis, MO, USA). Synthetic

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DNA templates (86–115 nt, sequences are shown in Table 1) were purchased

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from Eurofins Genomics (Tokyo, Japan).

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Preparation of digested amplicons

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The procedure of sample preparation is summarized in Figure 1. PCR reactions

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were performed on a 25 µL reaction scale and consisted of 0.625 U of Pfu-X

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DNA polymerase (Jena Bioscience, Jena, Germany), 0.2 mM of each dNTP

84

(Jena Bioscience), 1× detergent free Phusion HF buffer (New England Biolabs,

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Ipswich, MA, USA), either 12.5 ng of an extracted fish DNA or 0.5 pmol of the 5



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synthetic DNA template and 0.4 µM of each primer. Two forward primers

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(5′-CCATGTGGAATGAAAACACC-3′ for Takifugu and

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5′-AAAAACAAGAGCCACAGCTCTAA-3′ for Lagocephalus) and one reverse

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primer (5′-CCCTAGGGTAACTCGGTTCG-3′) were designed with the help of

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Primer3Plus software (http://www.bioinformatics.nl/primer3plus/) and

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synthesized by Fasmac (Atsugi, Japan). These primers were used to amplify

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114–115-bp and 86-bp amplicons of the mitochondrial 16S ribosomal RNA gene

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of Takifugu species and Lagocephalus species, respectively (Figure 1).25,26

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Thermocycling was performed in a T100 thermal cycler (Bio-Rad, Hercules, CA,

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USA) under the following conditions: 95 °C, 2 min, then 30 cycles at 95 °C, 30 s,

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56 °C, 30 s, 72 °C, 30 s, 72 °C, 7 min and 12 °C hold. After PCR, 2 µL of the 10×

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universal buffer M (Takara Bio, Otsu, Japan, containing 100 mM Tris-HCl (pH

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7.5), 100 mM MgCl2, 10 mM dithiothreitol and 500 mM NaCl) and 1 µL of each

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FastDigest restriction enzyme (Dra I and Msp I, Thermo Fisher Scientific) were

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added to the tube and incubated for 30 min at 37 °C, followed by additional

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incubation for 5 min at 72 °C. A portion of the reaction solution was analyzed by

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polyacrylamide gel electrophoresis (PAGE) using the staining dye Gel Green

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(Biotium, Hayward, CA, USA), if necessary. The remaining solution was filtered

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with a Nanosep MF centrifugal device containing a Bio-inert membrane (0.45 µm,

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Pall Corporation, Port Washington, NY, USA) before LC/ESI-MS analysis. The

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filtrates were stored at −20 °C until analysis.

107 108

LC/ESI-MS analysis 6


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The LC/ESI-MS was performed using an Ultimate 3000 liquid chromatograph

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and a Q Exactive quadrupole–Orbitrap hybrid mass spectrometer (Thermo

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Fisher Scientific). A reverse-phase silica monolith column (MonoBis C18, Kyoto

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Monotech, Kyoto, Japan; 2.0×50 mm, mesopore size 30 nm, product number

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2050H30ODS) was used for the separation at 20 °C with a MonoBis C18 guard

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column (Kyoto Monotech). The mobile phase, delivered at a flow rate of 0.4

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mL/min, was 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine

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(pH 7.9) (A)–methanol (B).27 The proportion of B in the mobile phase was

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changed over a linear gradient as follows: 0–0.5 min, 5%; 0.5–1 min, 5–30%; 1–

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3.5 min, 30–40%; 3.5–5 min, 40–98%; 5–6 min, 98%; 6–6.05 min, 98–5% and

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6.05–8 min, 5%. The sample injection volume was 1.0 µL except for the tests of

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the stability of mass accuracy and the temperature of the column (0.5 µL each).

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The post-column flow was connected to the mass spectrometer for 3.5–6 min

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after sample injection. A heated ESI probe (HESI-II, Thermo Fisher) was used in

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negative ion mode and with the following parameters: spray voltage, 2.5 kV;

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vaporizer temperature, 350 °C; sheath gas, 50 arbitrary units (a.u.); auxiliary gas,

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15 a.u. The parameters for the desolvation at the entrance of the mass

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spectrometer were as follows: Capillary temperature, 350 °C; sweep gas

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pressure, 1 a.u.; S-lens radiofrequency level, 100. High resolution mass analysis

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was performed using the following parameters: scan range, m/z 700–3500;

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nominal resolution (at m/z 200), 140,000; auto gain control target, 1×106;

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microscans, 3; maximum ion time, 50 ms; in-source collision induced

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dissociation, 15 eV. The C-trap pressure, which can be adjusted with the valve 7



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inside the chassis, was reduced to 0.2 MPa to improve the sensitivity for long

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oligonucleotides. A lock mass was not assigned. Mass calibration was

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performed daily using sodium trifluoroacetate clusters (n = 5–24, m/z

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792.85908–3376.38045).28 The calibration solution was 0.5 mg/mL sodium

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trifluoroacetate (pH 3.5) in water–acetonitrile (1:1, v/v) and was infused into the

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mass spectrometer at the flow rate of 10 µL/min. The in-source collision-induced

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dissociation voltage was set to 60 eV to develop the cluster ions at higher mass

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range.

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Peak detection and deconvolution from the raw data using Xtract algorithm29,30

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were carried out automatically with the Protein Deconvolution version 3.0

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software (Thermo Fisher). The parameters were as follows: fit factor 90%,

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charge range 3–50.

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Analysis of pufferfish and other fish species

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Twenty-nine pufferfish samples (1 boiled skin of T. rubripes, 3 raw livers and 1

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ovary of Takifugu porphyreus, 1 raw muscle tissue of Takifugu vermicularis, 2

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fresh whole bodies of Takifugu pardalis, 1 dried fillet of Takifugu snyderi, 19

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fresh whole bodies of Takifugu poecilonotus and 1 dried fillet of Lagocephalus

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wheeleri) and eight raw muscle tissues of other fish (Atlantic mackerel (Scomber

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scombrus), Japanese horse mackerel (Trachurus japonicus), Japanese pilchard

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(Sardinops melanostictus), Skipjack tuna (Katsuwonus pelamis), Yellowfin tuna

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(Thunnus albacares), Japanese amberjack (Seriola quinqueradiata), Atlantic 8


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salmon (Salmo salar) and Flying fish (Cypselurus pinnatibarbatus japonicus)

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were obtained from fish wholesalers and retail markets in Japan. Total genomic

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DNA in fish tissue (~50 mg) was extracted with a DNeasy Blood and Tissue Kit

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(Qiagen, Hilden, Germany) in accordance with the protocol of the manufacturer.

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In the case of whole body samples, fresh fins were collected for extraction. The

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species of the pufferfish samples being studied were verified using the official

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method of the Office of Import Food Safety, Inspection and Safety Division,

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Department of Food Safety, Ministry of Health, Labour and Welfare, Japan

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(2011), which was based on Sanger sequencing of the 16S ribosomal RNA

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gene.6 The same method was also used to confirm the common names of other

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fish samples with the aid of the BLAST search (DNA Data Bank of Japan).

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Amplification and LC/ESI-MS analysis were performed as described above.

167 168

RESULTS AND DISCUSSION

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Optimization of LC/ESI-MS conditions

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Past studies dealing with LC/ESI-MS analysis of long oligonucleotides have

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mostly been carried out using a poly(styrene-divinylbenzene) (PS-DVB) capillary

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monolith column (0.2 mm i.d.) at low flow rate (2 µL/min).18–24,31 Instead, we

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tested a narrow-bore type (2.0 mm i.d.) reverse-phase silica monolith column for

174

the separation of long oligonucleotides employing a much higher flow rate (400

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µL/min), which enabled the use of a heated sprayer at the interface. Accordingly,

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the DNA duplexes were denatured, regardless of column temperature, when the

9



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mobile phase was sprayed into the ion source with the aid of heated nitrogen

178

(350 °C).

179

Column temperature was optimized (Figure 2). Signals for the three pairs of

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DNA duplexes (26, 37 and 53 bp) and the primers were separated from one

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another at 20 °C and 60 °C, whereas distorted peaks were observed at 40 °C.

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These differences may be related to the formation of the double helix structure

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during chromatographic separation. Oberacher et al reported that a DNA duplex

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longer than 25–50 bp could be denatured in the analytical column at a

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temperature between 50–80 °C.24 Accordingly, possible explanations for this

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behavior are that the DNA duplexes maintained their rigid structure at 20 °C,

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denatured partially at 40 °C, and denatured completely at 60 °C. Taking the

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separation behavior and the maximum column temperature recommended by

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the manufacturer (40 °C) into consideration, the column temperature was

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adjusted to 20 °C. Despite the low column temperature, the maximum back

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pressure of the system (12.5 MPa) was below the upper limit of the column (30

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MPa) recommended by the manufacturer, owing to the inherently large channel

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size of the silica monolith.32

194 195

Discrimination of synthetic DNA by PCR-RFLP coupled with LC/ESI-MS

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The PCR-RFLP method was used to discriminate 11 synthetic DNA (range, 86

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to 115 bp) samples (Table 1), which served as the positive control templates for

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the pufferfish. The list for the target pufferfish was compatible with the published

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method by the Japanese government based on the Sanger sequencing. The 10


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exceptions to this were T. pardalis and T. snyderi and, given that the target

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sequences for these two species are identical, T. pardalis was selected as being

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a representative example. The result obtained by PAGE is shown in Figure 3a,

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and that obtained by LC/ESI-MS is given in Figure 3b and Table 2. Using PAGE,

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the band pattern could only be divided into three groups: Takifugu chrysops,

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another species of genus Takifugu and genus Lagocephalus (Figure 3a). Each

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band, which corresponded to the DNA duplex detected by PAGE, was also

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detected by LC/ESI-MS as a peak (Figure 3b), and where each peak in the

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chromatogram represented the mass spectra of the forward and reverse

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oligonucleotides. Because the digestion with the endonucleases was performed

210

just after PCR amplification without purification, the sticky end generated by the

211

Msp I endonuclease was filled up with the remaining DNA polymerase during

212

incubation (Figure 1).12

213 214

Method validation

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Accuracy and precision data for the LC/ESI-MS analyses are presented in Table

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2. Although an internal mass standard was not used, excellent accuracy (mass

217

error, 0.83 ppm on average) and precision (standard deviation, 0.49 ppm on

218

average) were achieved. Although this performance reflects the inherent

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performance of the Orbitrap analyzer, deconvolution also contributed to an

220

improved accuracy and precision. Because dozens of peaks having different

221

charge states and isotopic compositions are taken into account for the

222

calculation of a monoisotopic mass, the monoisotopic mass calculated by 11



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deconvolution has a reduced observational error and must have been more

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accurate than any molecular mass information determined from an individual

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peak.30,33 In addition, the use of the monoisotopic mass for mass

226

characterization is preferable to that of the average mass for achieving

227

enhanced accuracy and precision.33 Because the monoisotopic mass is, for all

228

practical purposes, independent of the isotope ratio,34 the acquired data can be

229

directly compared with the theoretical monoisotopic mass calculated from the

230

molecular formula. An alternative mass spectrometry metric for a biomolecule is

231

the average mass, which is defined as the centroid of the isotopic distribution of

232

the biomolecule. However, according to a calculation by Zubarev et al, the

233

accuracy of the average mass measurement is limited to 10 ppm for

234

biomolecules larger than 10,000 Da because of natural variations of isotopic

235

abundances of the elements. Furthermore, the non-symmetric shape of the

236

isotopic distribution causes negative bias.33 Manduzio et al reported an average

237

mass deviation of 9.15 ± 7.11 ppm as a result of analyzing a 114-bp PCR

238

product despite an Orbitrap analyzer being used.12 Kullolli et al used an

239

LTQ-Orbitrap Veros for analyzing microRNA ranging from 22 to 25 nt.35 A mass

240

accuracy of 1.3 ppm on average was reported; however, these studies

241

compared the monoisotopic peaks of the multiply charged ions with the

242

corresponding theoretical ones instead of the deconvoluted monoisotopic mass

243

of the molecule. Because the monoisotopic peak disappears from a mass

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spectrum with increasing molecular mass,33 the approach involving the

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monoisotopic peaks is only applicable for short oligonucleotides. 12


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Periodic analyses were carried out to evaluate the stability of mass accuracy.

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Digested amplicons derived from the synthetic DNA of Takifugu chrysops were

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analyzed every 10 h for 180 h. As a result, all the monoisotopic masses of the

249

digested PCR amplicons were successfully determined within a mass error of

250

2.8 ppm without any additional mass calibration. Consequently, a daily

251

calibration can be replaced by a weekly calibration.

252

As a result of the above findings and observations, a flow chart for the

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discrimination of the pufferfish is proposed (Figure 4). The target pufferfish

254

always yields two or three peaks in the TIC chromatogram at a retention time of

255

4–5.5 min; otherwise the sample is not the target pufferfish. If two peaks are

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observed in the range, a pair of the monoisotopic masses of the DNA duplex

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corresponding to the latter peak will be compared to the theoretical values to

258

determine if any one of the species is T. chrysops, L. inermis, L. gloveri, L.

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lunaris or L. wheeleri. The mass tolerance for the determination is 3 ppm which

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does not cause overlap. If three peaks are observed in the range, a similar

261

matching procedure is required for the second peak in some cases as well as for

262

the third peak (Figure 4).

263 264

Mixed template

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The synthetic DNA template of Lagocephalus lunaris was mixed with that of L.

266

wheeleri at ratios of 1:1, 1:4 and 1:19, and the mixtures were analyzed as

267

described above. As the result of the preparation, four pairs of DNA duplexes

268

would have existed in the sample solution and the success of the discrimination 13



269

relies on the difference of the masses between each oligonucleotide. The results

270

are shown in Figure 5. In the case when the mass difference was about 25 Da

271

(Figure 5a, single base substitution) and 7 Da (Figure 5b, triple base

272

substitution), each strand could be analyzed independently for the mixing ratio of

273

1:1; however, minor constituents could not be calculated properly. When the

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difference was about 6 Da (Figure 5c, the complementary sequence of Figure 5b)

275

because of an overlapping of clusters of peaks, the isotopic mass could not

276

calculated even for a mixing ratio of 1:1 although this could be compensated for

277

by the results for the complementary strand. These observations are basically

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consistent with the study of Manduzio et al who investigated the discrimination

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ability for an equal mixture of templates with single base substitution.12 They

280

reported that a mass difference of 16 Da (A G) could be discriminated,

281

although that of 9 Da (T A) could not be discriminated for analysis of 114 bp

282

DNA. This was overcome by emphasizing the difference of the m/z values using

283

endonuclease digestion, which is consistent with the present strategy.

284

As an alternative to calculating the monoisotopic mass, the extracted ion

285

chromatograms (XICs) of the most abundant isotopic ions were examined as a

286

means to discriminate T. rubripes from T. pardalis. However, it was impossible to

287

discriminate the two species using XIC because of the overlapping of the peaks

288

(Figure 6), although they could be discriminated using a deconvolution approach

289

(Table 2). Therefore, deconvolution was the preferred approach for species

290

identification, especially when the sample was considered to be derived from a

291

single source. 14


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292 293

Application for small samples

294

A small amount of pufferfish alone or its presence in another fish was analyzed

295

by the present methods. As a result of the analysis of a raw muscle tissue

296

homogenate of T. vermicularis (0.1 mg equivalent, n = 2), all the expected DNA

297

fragments were successfully identified with an average mass error of 1.50 ppm.

298

In addition, raw muscle tissue of T. vermicularis mixed with that of Korean black

299

scraper (Thamnaconus modestus) (2.5 wt % of T. vermicularis in 50 mg mixture)

300

could be identified with an average mass error of 1.15 ppm (n = 2). Therefore,

301

the present method is considered applicable to small sample amounts as long as

302

PCR amplification proceeds successfully.

303 304

Discrimination of pufferfish and other fish samples

305

The present method was applied to 29 pufferfish samples and 8 other fish

306

species (Figure 7). The results for discrimination based on the present criteria

307

(Figure 4) were consistent with the official method based on Sanger sequencing,

308

although the present method could not discriminate T. pardalis from T. snyderi

309

because these species contain the same amplicon sequence (data not shown).

310

Additionally, no deconvolution value was calculated from the chromatograms of

311

fish samples other than pufferfish.

312

As mentioned above, the use of LC/ESI-MS for fish genotyping has been

313

demonstrated for the first time. A remaining problem is the interpretation of data

314

obtained from a mixed template at a closer ratio as mentioned above. Using 15



315

multiplex PCR with another pair of pufferfish-specific or species-specific primers

316

may be an effective way for resolving analytes in a mixture.

317 318

Funding

319

This work was supported by a Grant-in-Aid for Scientific Research by the Japan

320

Society for the Promotion of Science (15K08060).

321

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Distribution of tetrodotoxin in pufferfish collected from coastal waters of

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Sihanouk Ville, Cambodia. Shokuhin Eiseigaku Zasshi 2008, 49, 361–365.

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White, K. D.; Thompson, T. M.; Wahl, M.; Pham, T. D.; Guichard, F. M.; Huh, I.;

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Hwang, D.-F., Puffer fish-based commercial fraud identification in a segment of

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assay for the detection of pufferfish products. J. Food Prot. 2010, 73, 1698–

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22


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446

FIGURE CAPTIONS

447

Figure 1. Method summary. Bold indicates the nucleotides filled up with the

448

remaining DNA polymerase. p indicates a phosphate group.

449

Figure 2. Chromatograms of the PCR products of the synthetic DNA of Takifugu

450

rubripes for comparison of temperatures for the MonoBis C18 column. TIC, total

451

ion current.

452

Figure 3. Results for PCR-RFLP analysis of synthetic pufferfish DNA. a, PAGE;

453

b, LC/ESI-MS.

454

Figure 4. Flow chart for the discrimination of pufferfish. TIC, total ion current. RT,

455

retention time.

456

Figure 5. Total ion current chromatograms obtained from the mixed templates.

457

nt, nucleotides.

458

Figure 6. An example of failure of discrimination between T. rubripes and T.

459

pardalis using individual isotopic peaks. nt, nucleotides.

460

Figure 7. Total ion current chromatograms derived from actual fish samples.

461

23



Table 1

Page 24 of 33

DNA Sequences Used for the PCR Templates.

Species

DNA sequencesa

Takifugu rubripes Takifugu porphyreus

CCATGTGGAATGAAAACACCCTTTTTAAACCCAAGAGTCACCACTCTAGGATACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

Length (nt)b 114

Refer encec (26)

Accession No. AP006045

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAGGATACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009529

Takifugu chrysops

CCATGTGGAATGAAGACACCCTCTCTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009525

Takifugu vermicularis

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAATTAATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

115

–

AB741999

Takifugu pardalis

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009528

Takifugu niphobles

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAAT-AATGATCCGGCTTAAAGCCGATTAACGAACCGAGTTACCCTAGGG

115

(26)

AP009526

Takifugu poecilonotus

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAGT-AATGATCCGGCTT-AAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009539 AP009538

Lagocephalus wheeleri

AAAAAC-AAGAGCCACAGCTCTAATGAGCAGAACATCTGACCTACCA--GATCCGGC--ATAGCCGATCAACGAACCGAGTTACCCTAGGG

86

(26)

Lagocephalus gloveri

AAAAAC-AAGAGCCACAGCTCTAATGAACAGAACATCTGACCTACCA--GATCCGGC--ATAGCCGATCAACGAACCGAGTTACCCTAGGG

86

–

AB742032

Lagocephalus lunaris

AAAAAC-AAGAGCCACAGCTCTAATAAACAGAACATCTGACCAGCCA--GATCCGGC--CTAGCCGATCAACGAACCGAGTTACCCTAGGG

86

(25)

Not available

Lagocephalus inermis

AAAAAC-AAGAGCCACAGCTCTAATAAACAGAACATCTGACCCACCA--GATCCGGC--ACAGCCGATCAACGAACCGAGTTACCCTAGGG

86

–

AB742030

a

Underline indicates primer-binding site and bold indicates the recognition sites of the restriction enzymes. bnt, nucleotide. c–, unpublished.

24


Page 25 of 33


Table 2

LC/ESI-MS Analysis of the Amplicons Digested with the Endonucleases.

Species

T. rubripes

T. porphyreus

T. chrysops

T. vermicularis

T. pardalis

T. niphobles

T. poecilonotus

L. wheeleri

L. gloveri

L. lunaris

L. inermis

F/Ra

F F F R R R F F F R R R F F R R F F F R R R F F F R R R F F F R R R F F F R R R F F R R F F R R F F R R F F R R

Length (nt)b

26 53 37 37 53 26 26 53 37 37 53 26 79 37 37 79 26 54 37 37 54 26 26 53 37 37 53 26 26 53 38 38 53 26 26 53 37 37 53 26 52 36 52 36 52 36 52 36 52 36 52 36 52 36 52 36

Expected molecular Monoisotopic mass, n = 5 (interday) formula Theoretical Measured value Relative value (Da) (Da, average ± standard standard deviation) deviation (ppm) C254H321O154N94P25 7925.362 7925.366 ± 0.003 0.37 C515H647O307N208P53 16296.750 16296.763 ± 0.006 0.34 11466.909 11466.920 ± 0.002 0.18 C360H452O218N147P37 C360H456O225N132P36 11341.885 11341.890 ± 0.003 0.25 C522H659O331N186P53 16468.655 16468.661 ± 0.004 0.25 8085.340 8085.352 ± 0.001 0.13 C256H322O157N98P26 C254H321O154N94P25 7925.362 7925.367 ± 0.004 0.44 C516H647O306N210P53 16320.762 16320.764 ± 0.017 1.05 C360H452O218N147P37 11466.909 11466.919 ± 0.006 0.50 C360H456O225N132P36 11341.885 11341.892 ± 0.004 0.33 C522H660O332N183P53 16443.648 16443.651 ± 0.010 0.62 C256H322O157N98P26 8085.340 8085.350 ± 0.002 0.28 C768H965O458N303P78 24173.107 24173.129 ± 0.017 0.70 C360H452O218N147P37 11466.909 11466.918 ± 0.006 0.56 C360H456O225N132P36 11341.885 11341.891 ± 0.006 0.52 C779H980O489N283P79 24566.979 24567.021 ± 0.016 0.64 C254H321O154N94P25 7925.362 7925.367 ± 0.004 0.46 C526H661O314N209P54 16599.801 16599.821 ± 0.009 0.56 11466.909 11466.922 ± 0.006 0.49 C360H452O218N147P37 C360H456O225N132P36 11341.885 11341.890 ± 0.005 0.48 C533H672O336N190P54 16780.717 16780.724 ± 0.004 0.21 C256H322O157N98P26 8085.340 8085.349 ± 0.005 0.65 7925.362 7925.367 ± 0.002 0.30 C254H321O154N94P25 C516H648O307N207P53 16295.755 16295.764 ± 0.020 1.21 C360H452O218N147P37 11466.909 11466.922 ± 0.007 0.60 11341.885 11341.889 ± 0.006 0.49 C360H456O225N132P36 C523H660O331N185P53 16467.659 16467.671 ± 0.011 0.66 8085.340 8085.350 ± 0.003 0.36 C256H322O157N98P26 C254H321O154N94P25 7925.362 7925.367 ± 0.005 0.64 C516H648O307N207P53 16295.755 16295.764 ± 0.012 0.76 C370H465O225N149P38 11770.955 11770.971 ± 0.005 0.41 11654.943 11654.951 ± 0.004 0.30 C370H468O230N137P37 16467.659 16467.666 ± 0.006 0.34 C523H660O331N185P53 C256H322O157N98P26 8085.340 8085.353 ± 0.003 0.38 C254H321O154N94P25 7925.362 7925.369 ± 0.005 0.61 16311.750 16311.730 ± 0.014 0.84 C516H648O308N207P53 C360H453O220N144P37 11457.898 11457.910 ± 0.003 0.23 C360H455O223N135P36 11350.897 11350.892 ± 0.007 0.62 16452.660 16452.664 ± 0.007 0.45 C522H659O330N186P53 C256H322O157N98P26 8085.340 8085.349 ± 0.003 0.33 15937.744 15937.761 ± 0.008 0.48 C505H632O296N209P51 11138.852 11138.844 ± 0.010 0.87 C349H439O212N143P36 C511H647O327N179P52 16131.586 16131.595 ± 0.006 0.39 C350H443O219N130P35 11053.834 11053.844 ± 0.004 0.38 C505H632O295N209P51 15921.750 15921.765 ± 0.007 0.46 C349H439O212N143P36 11138.852 11138.848 ± 0.006 0.50 C512H648O328N178P52 16146.585 16146.580 ± 0.009 0.54 C350H443O219N130P35 11053.834 11053.849 ± 0.010 0.89 C505H631O293N212P51 15930.761 15930.776 ± 0.006 0.36 C348H439O213N141P36 11114.841 11114.846 ± 0.006 0.58 C512H649O330N175P52 16137.574 16137.583 ± 0.007 0.42 C350H442O218N133P35 11078.841 11078.836 ± 0.003 0.24 C504H631O293N210P51 15890.755 15890.773 ± 0.006 0.38 C348H438O211N144P36 11123.852 11123.851 ± 0.004 0.35 C513H649O330N177P52 16177.580 16177.590 ± 0.005 0.33 C350H443O220N130P35 11069.829 11069.836 ± 0.008 0.71

a

Average mass errorc (ppm) 0.57 0.80 0.93 0.45 0.42 1.44 0.73 0.78 0.85 0.60 0.53 1.19 0.92 0.84 0.58 1.69 0.69 1.21 1.11 0.42 0.40 1.06 0.64 1.11 1.08 0.51 0.76 1.21 0.82 0.80 1.36 0.74 0.39 1.57 0.91 1.21 1.10 0.53 0.42 1.07 1.04 0.90 0.61 0.88 0.94 0.49 0.51 1.33 0.96 0.51 0.59 0.37 1.10 0.26 0.64 0.84

F, forward strand; R, reverse strand (see Figure 1). bnt, nucleotide. cMass error is defined as the absolute deviation of a measured monoisotopic mass from the theoretical one.

25



Fish sample

DNA extraction

PCR

Enzymatic digestion

LC/ ESI-MS

Data analysis

Genus Takifugu (except for Takifugu chrysops) Forward primer for Takifugu 5ʹ 3ʹ 26 bp

Dra I digestion

Msp I digestion

TTT pAAA AAAp TTT

CCG pCGG GGCp GCC

53–54 bp

37–38 bp

3ʹ 5ʹ

Reverse primer

Takifugu chrysops Forward primer for Takifugu 5ʹ 3ʹ

Msp I digestion CCG pCGG GGCp GCC

TCTAAA AGATTT

37 bp

3ʹ 5ʹ

Reverse primer

79 bp

Genus Lagocephalus Forward primer for Lagocephalus

Msp I digestion CCG pCGG GGCp GCC

5ʹ 3ʹ 52 bp

36 bp

3ʹ 5ʹ

Reverse primer

Figure 1

26


Page 26 of 33

Page 27 of 33


Relative intensity

20 °C

60 °C

40 °C

1.04 ×107

4.53 ×106

1.08 ×107

0 1.06 ×105

0 2.43 ×104

0 7.86 ×104

26 bp

0 6.80 ×104 37 bp 0 7.43 ×104

53 bp

0 6.50 ×104

m/z = 2835.720 + 2866.976

37 bp

37 bp 0 2.52 ×104

0 8.45 ×104 53 bp

53 bp 0 6

m/z = 2641.785 + 2695.111

26 bp

26 bp 0 2.04 ×104

0 3

TIC

m/z = 1163.548 + 1175.827

0 3

6

3

6

Time / min

Figure 2

27



Figure 3

28


Page 28 of 33

Page 29 of 33


Draw TIC chromatogram

≤1

Peak Number at RT = 4–5.5 min

3

Negative

(genus Takifugu) 2 (Takifugu chrysops or genus Lagocephalus)

Monoisotopic mass of the 3rd peak(Da)

Monoisotopic mass of the 2nd peak (Da)

16443.599–16443.697 16452.610–16452.709 16467.610–16467.709 16468.605–16468.704

T. poecilonotus T. exascurusa

16400

6

16500 16599.751–16599.851 16780.667–16780.767

16200

T. vermicularis

16131.537–16131.634 16137.525–16137.622 16146.537–16146.634 16177.531–16177.629

T. chrysops

24173.034–24173.179 24566.905–24567.053

T. porphyreus T. obscurusa

L. lunaris

L. gloveri

L. wheeleri

15890.707–15890.803 15921.702–15921.797 15930.713–15930.809 15937.697–15937.792

16000

16100

16300

6

L. inermis

15900

5 Time / min

5 Time / min

T. rubripes

4

4

16295.706–16295.804 16296.701–16296.799 16311.701–16311.799 16320.713–16320.811

Monoisotopic mass of the 2nd peak (Da)

4

11300

5 Time / min

11466.875–11466.944 11500 11600

11770.920–11770.991 11800

Figure 4

29


T. niphobles

11654.908–11654.978 11700

T. pardalis T. snyderi T. ocellatusa T. xanthopterusa T. stictonotusa

11341.851–11341.919 11400

6


a 36 nt, reverse strand [M-4H]4-, ∆ = 25.006 (Da)

L. wheeleri : L. lunaris

L. wheeleri 0.48 ppm

b 52 nt, forward strand [M-5H]5- , ∆ = 6.983 (Da) L. lunaris 0.29 ppm


L. lunaris 0.70 ppm

4:1


19:1

2760

L. wheeleri NA


L. lunaris NA



L. lunaris NA

2770

c 52 nt, reverse strand [M-5H]5- , ∆ = 5.988 (Da)


L. lunaris 0.44 ppm

1:1


L. lunaris NA

3180

3190

Figure 5

30


Page 30 of 33

3220

L. lunaris NA

L. lunaris NA

L. lunaris NA

3230

m/z

Page 31 of 33


Figure 6

31


Relative intensity


2.39×106

Negative control

0 3.60×107

T. rubripes

0 4.11×107

T. porphyreus

0 3.95×107

T. vermicularis

0 3.65×107

T. pardalis

0 1.63×107

T. snyderi

0 3.55×107

T. poecilonotus

0 3.07×107

L. wheeleri

0 6.17×106

Atlantic mackerel (Scomber scombrus)

0 4.15×106 0 4.63×106 0 2.09×106 0 1.86×106

0 2.67×106

Japanese horse mackerel (Trachurus japonicus)

Japanese pilchard (Sardinops melanostictus)

Skipjack tuna (Katsuwonus pelamis)

Yellowfin tuna (Thunnus albacares)

Japanese amberjack (Seriola quinqueradiata)

0 6.51×106

Atlantic salmon (Salmo salar)

0 2.81×106

Flying fish (Cypselurus pinnatibarbatus japonicus)

0

3.5

4.0

4.5 5.0 Time / min

5.5

6.0

Figure 7

32


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Graphic for TOC

33


Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP

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