线粒体细胞色素b基因在鱼唇制品物种鉴定中适用性的研究

目的 探讨线粒体细胞色素b基因(cytochrome b, Cyt b)作为DNA条形码在鱼唇制品物种鉴定中的适用性。方法 对全国31个城市购买的252份鱼唇样品进行聚合酶链式反应(polymerase chain reaction, PCR)测序,同源基因比较分析,构建系统发育树,鉴定制作鱼唇产品的鱼种,并对其进行濒危评价分析。结果 成功鉴定250个样品,一致性物种基因序列相似性在99%以上,涉及8个鲨鱼物种,最多样品为大青鲨(Prionace glauca),占样品65.5%,其余还有镰形真鲨(Carcharhinus falciformis)、路氏双髻鲨(Sphyrna lewini)、锤头双髻鲨(Sphyrna zygaena)等7类鲨鱼物种。结论 Cyt b可以作为对鲨鱼物种进行鉴定的一种DNA条形码,在对鲨鱼种鉴定时可以使用Cyt b基因及细胞色素氧化酶亚基I基因联合鉴定条形码,为深加工海产品物种鉴定提供更多的技术支撑。     

Studies on Aspergillus section Flavi isolated from maize in northern Italy

In 2003, for the first time in Italy, significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM(1) at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 degrees C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 degrees C, while AFB(1) production was optimal at 25 degrees C. Regarding water availability (water activity, a(w)), 0.99 a(w) was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 a(w)) was AFB(1). This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.     

Biodiversity of Aspergillus section Flavi in the United States: A review

Fungi belonging to Aspergillus section Flavi are of great economic importance in the United States due to their ability to produce toxic and carcinogenic aflatoxins in agricultural commodities. Development of control strategies against A. flavus and A. parasiticus, the major aflatoxin-producing species, is dependent upon a basic understanding of their diversity in agricultural ecosystems. This review summarizes our current knowledge of species and population diversity in the United States in relation to morphology, mycotoxin production and genetic characters. The high genetic diversity in populations of aflatoxigenic fungi is a reflection of their versatile habits in nature, which include saprotrophic colonization of plant debris in soil and parasitism of seeds and grain. Genetic variation within populations may originate from a cryptic sexual state. The advent of intensive monoculture agriculture not only increases population size but also may introduce positive selective pressure for aflatoxin production due to its link with pathogenicity in crops. Important goals in population research are to determine how section Flavi diversity in agricultural ecosystems is changing and to measure the direction of this evolution.     

A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds

Thirty-five samples of poultry feeds and corresponding raw materials (maize, soybean and meat meal) from a processing plant were analyzed to evaluate the distribution and toxigenicity of Aspergillus section Flavi isolates. Mycological analysis of the samples indicated the presence of five fungal genera (Aspergillus, Penicillium, Fusarium, Cladosporium, and Eurotium). Aspergillus flavus was the predominant species being present in 48.5% of the analyzed samples. Ninety-one isolates belonging to Aspergillus section Flavi were isolated; ninety were identified as A. flavus and only one as A. parasiticus. Fifty-seven isolates were capable of producing sclerotia, 41 were identified as L-type strains and 16 as type S. Fifty-seven percent of the isolates produced AFB₁ levels ranging from 0.05 μg/kg to 27.7 μg/kg whereas 86.8% produced CPA from 1.5 μg/kg to 137.8 μg/kg. L-strains produced from 0.05 to 14.8 μg/kg of aflatoxin and type S produced levels from 0.05 to 1.65 μg/kg. No significant differences in CPA production among S- and L-strains were observed. Sclerotial isolates produced AFB₁ levels ranging between 0.05 and 27.7 μg/kg and CPA levels from 3.8 to 47.3 μg/kg. More than half of the A. flavus isolates were able to produce AFB and CPA simultaneously. Twenty percent of the 35 samples were contaminated with aflatoxin B₁ whereas 34.3% were contaminated with CPA. The high rate of CPA producing isolates represents a potential risk of contamination with this toxin in poultry feeds.     

Polyphasic characterization of Aspergillus section Flavi isolated from animal feeds in Algeria

In Algeria, little information is available on the population structure of Aspergillus section Flavi in raw materials and resultant animal feeds. A total of 172 isolates belonging to Aspergillus section Flavi were recovered from 57 animal feeds and identified on the basis of macro and micro-morphological characters, mycotoxin production and genetic relatedness. For the molecular analysis, sequencing of the calmodulin gene (CaM) and the internal transcribed spacer (ITS) regions were performed for representative isolates. Four distinct morphotypes were distinguished: Aspergillus flavus (78.5%), Aspergillus tamarii (19.2%), Aspergillus parasiticus (1.7%), and Aspergillus alliaceus (0.6%). All A. flavus isolates were of the L type and no correlation between sclerotia production and aflatoxigenicity was observed. Our results showed that 68% of the A. flavus strains produced aflatoxins B (AFB), and 72.7% were cyclopiazonic acid (CPA) producers. The three isolates of A. parasiticus were able to produce AFB and aflatoxins G but not CPA whereas, all the strains of A. tamarii produced only CPA. The obtained results revealed the presence of different species of Aspergillus section Flavi, among which were aflatoxin producers. This study provides evidence useful for considerations in aflatoxin control strategies.     

Aspergillus section Flavi and aflatoxins in dried figs and nuts in Algeria

The presence of Aspergillus section Flavi and aflatoxin (AF) contamination was investigated in 112 samples of peanuts, almonds and dried figs collected in Algeria. The occurrence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in different commodities has been determined with a sensitive method based on high performance liquid chromatography (HPLC) coupled with fluorescence detection with post-column photochemical derivatisation. Analytical results indicated that 28 samples of peanuts, 16 samples of almonds and 26 samples of dried figs contained detectable levels of AFs. A total of 69 samples (61.6%) were contaminated with AFB1 ranging from the limit of quantification to 174 µg kg^?1. AFB2 was found in 12 samples (10.7%) and varied from 0.18 to 193 µg kg^?1. Seven samples revealed AF concentrations lower than the limit of quantification. Eleven peanut and fourteen dried fig samples exceeded the European maximum limits for AFB1.     

Identifying unknown game species: experience with nucleotide sequencing of the mitochondrial cytochrome b gene and a subsequent basic local alignment search tool search

 Game meat is often a target for fraudulent labelling. Polymerase chain reaction (PCR) analysis on the mitochondrial cytochrome b sequence with subsequent restriction fragment length polymorphism is the most widely used method of identifying meat species. The lack of reference meat species and the possibility of point mutations affecting the typical restriction pattern of a species sometimes results in an analytical uncertainty. Nowadays, sequencing of the PCR fragment with a subsequent search in an internet-accessible database can avoid these problems. The database search results in a list of sequences in order of the highest percentage of correspondence. In this work it is shown what correspondences within different vertebrate classes, orders, families and species can be expected. Classing with the correct genus is possible, at least for game meat. Received: 20 July 2000 / Revised: 4 October 2000     

Updated profile of aflatoxin and Aspergillus section Flavi contamination in rice and its byproducts from the Philippines.

Our goal was to develop an updated profile of aflatoxin (AF) and AF-producing fungi contamination in rice and its byproducts from the Philippines. The total AF levels in 78 samples of polished and brown rice, determined by an immunoaffinity column clean-up method coupled with HPLC (detection limit: 25 ng/kg), ranged from <0.025-2.7 microg/kg (mean of positive samples: 0.37 microg/kg) and 0.03-8.7 microg/kg (mean of positive samples: 2.7 microg/kg), respectively. The incidence (% of positive samples) of AF in polished and brown rice were 94% and 100%, respectively. The AF levels in polished rice imported from Thailand and Vietnam were approximately 20% of the levels found in locally produced polished rice. AF levels decreased as the rice progressed through the various stages in milling. Fungi recovered include toxigenic Aspergillus flavus and A. parasiticus with an incidence ranging from 14% in rice bran to 78% in rough rice and producing <0.025-6200 microg/kg total AF in in vitro cultures on rice. All samples of rice bran and rice hull contained AF at levels ranging from 0.27-11 microg/kg. The estimated potential daily intake of AFB(1) from rice is between 0.1 and 7.5 ng/kg of body weight/day, the mean of which is 1.0 ng representing 9.1-5.3 times the estimated tolerable daily intake for AFB(1) reported to date for Asia. Thus, Filipinos have a potentially high risk of exposure to AF that can be easily controlled through proper post-harvest handling and storage of rice and its byproducts.     

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines were examined. The average mold counts of rough rice, brown rice, and locally produced polished rice were 4.1 x 10(3), 1.0 x 10(3), and 1.1 x 10(3) CFU/g, respectively. Average Aspergillus section Flavi counts of the same samples were 3.0 x 10(2), 1.1 x 10(2), and 2.6 x 10(2) CFU/g, respectively. Twenty-seven percent of mold isolates from rough rice, polished rice, and brown rice were section Flavi spp., 31% of which were toxigenic. No section Flavi isolates were obtained from imported rice samples from Thailand and Vietnam. Aspergillus section Flavi was also isolated from rice hull, rice bran, and settled dust from rice milling operations. Toxigenic isolates of both Aspergillus flavus and Aspergillus parasiticus were present in at least one sample of each type of rice and rice by-product except settled dust. Aflatoxins produced in vitro by the isolates ranged from <1 microg/kg to 6,227 microg/kg. A. flavus isolates produced only B aflatoxins, whereas A. parasiticus isolates produced both B and G aflatoxins. Although total mold counts of Philippine rice and its by-products are within tolerable limits, the establishment of maximum limits in counts of potentially aflatoxigenic species in foods and feeds is important because the mere presence of toxin producers is considered a possible risk factor. The results of this research illustrate the need for strict monitoring of rice during both storage and marketing, especially in warm and humid seasons when infestation and consequent production of aflatoxins by Aspergillus section Flavi is expected.     

Aspergillus species from section Flavi isolated from soil at planting and harvest time in peanut‐growing regions of Argentina

Aspergillus species from section Flavi were isolated from soil samples in three peanut‐growing regions of Córdoba Province, Argentina. The samples were collected during the planting and harvest periods. Both total fungal population and Aspergillus species from section Flavi showed no significant differences between planting and harvest time in two of the regions evaluated. Only in one region were there significant differences in cfu g^?1 of total fungal population and Aspergillus species from section Flavi. A flavus was the dominant species isolated in all three localities during the planting and harvest periods. There were significant differences (p < 0.05) in the ratio of toxigenic and atoxigenic strains dependent on the period and the region evaluated. In one region, higher frequencies of toxigenic A flavus and A parasiticus in soil were found and a high contamination level of aflatoxins was detected in peanut seeds. Copyright © 2003 Society of Chemical Industry     

Impact of volatile compounds generated by essential oils on Aspergillus section Flavi growth parameters and aflatoxin accumulation

BACKGROUND: The advantage of essential oils is their bioactivity in the vapour phase, a characteristic that makes them attractive as possible fumigants for stored grain protection. In this study the antifungal and antiaflatoxigenic effects of the volatile fractions of five essential oils (EOs) were evaluated by vapour contact on Aspergillus section Flavi isolates. RESULTS: In maize meal extract agar the volatile fractions of Pimpinella anisum L. (anise), Pëumus boldus Mol. (boldus), Hedeoma multiflora Benth. (mountain thyme), Lippia turbinata var. integrifolia (Griseb.) (poleo) and Syzygium aromaticum L. (clove) were able to decrease the growth rate and lag phase of aflatoxigenic isolates. Boldus EO showed the best antifungal effect on Aspergillus section Flavi growth rate. In sterilised maize grains, boldus and poleo EOs showed antifungal effects on growth rate and aflatoxin accumulation. The volatile fraction of boldus EO completely inhibited the growth of isolates at water activity (a_w) levels of 0.955, 0.930 and 0.900, while poleo EO showed this effect only at the lower a_w levels (0.930 and 0.900). All aflatoxigenic isolates showed reduced total aflatoxin accumulation in the presence of boldus EO under all a_w conditions. CONCLUSION: These findings clearly indicate that the volatile fraction of boldus EO could be used to control aflatoxigenic fungi in stored maize. Copyright © 2009 Society of Chemical Industry     

Identification of meat species by PCR-RFLP of the mitochondrial COI gene

Meat authenticity verification is pertinent for economical, religious or public health concerns. The present study investigates the use of PCR-RFLP of a part of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene for identification of species origin of raw meat samples of cow, chicken, turkey, sheep, pig, buffalo, camel and donkey. PCR yielded a 710-bp fragment in all species. The amplicons were digested with seven restriction endonucleases (Hind II, Ava II, Rsa I, Taq I, Hpa II, Tru 1I and Xba I) that were selected based on the preliminary in silico analysis. Different levels of polymorphism were detected among samples. The level of COI variation revealed using only Hpa II was sufficient to generate easily analyzable species-specific restriction profiles that could distinguish unambiguously all targeted species. Compared to previously published reports for the determination of meat origin at the molecular level, the approach developed here is much cheaper and faster for routine identification of meats in food control laboratories.     

Application of essential oils in maize grain: impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation

The antifungal activity of Pimpinella anisum L. (anise), Pëumus boldus Mol (boldus), Hedeoma multiflora Benth (mountain thyme), Syzygium aromaticum L. (clove), and Lippia turbinate var. integrifolia (griseb) (poleo) essential oils (EOs) against Aspergillus section Flavi was evaluated in sterile maize grain under different water activity (a_w) condition (0.982, 0.955, and 0.90). The effect of EOs added to maize grains on growth rate, lag phase, and aflatoxin B₁ (AFB₁) accumulation of Aspergillus section Flavi were evaluated at different water activity conditions. The five EOs analyzed have been shown to influence lag phase and growth rate. Their efficacy depended mainly on the essential oil concentrations and substrate water activity conditions. All EOs showed significant impact on AFB₁ accumulation. This effect was closely dependent on the water activity, concentration, and incubation periods. Important reduction of AFB₁ accumulation was observed in the majority of EO treatments at 11 days of incubation. Boldus, poleo, and mountain thyme EO completely inhibited AFB₁ at 2000 and 3000 μg g⁻¹. Inhibition of AFB₁ accumulation was also observed when aflatoxigenic isolates grew with different concentration of EOs during 35 days.     

Screening of Argentine plant extracts: impact on growth parameters and aflatoxin B1 accumulation by Aspergillus section Flavi

The effect of essential oils, ethanolic and aqueous extract of 41 vegetable species on Aspergillus section Flavi growth was evaluated. The in vitro screen was a two-stage process. A wide-spectrum initial screen which identified promising antifungal plant extracts was carried out first. After that, identified extracts were studied in more detail by in vitro assays. A total of 96 plant extracts were screened. Essential oils were found to be the most effective extract controlling aflatoxigenic strains. Clove, mountain thyme, poleo and eucalyptus essential oils were selected to study their antifungal effect. Studies on percentage of germination, germ-tube elongation rate, growth rate, and aflatoxin B1 accumulation were carried out. Clove, mountain thyme and poleo essential oils showed the most antifungal effect under all growth parameters analyzed as well as aflatoxin B1 accumulation. Our results suggest that mountain thyme and poleo, which are native vegetal species of Argentina, and clove essential oils could be used alone or in conjunction with other substances to control the presence of aflatoxigenic fungi in stored maize.     

Increase in aflatoxins due to Aspergillus section Flavi multiplication during the aerobic deterioration of corn silage treated with different bacteria inocula

The growth of Aspergillus flavus and the production of aflatoxins (AF) during the aerobic deterioration of corn silage represent a problem for animal and human health. This experiment was conducted to evaluate whether the presence of A. flavus and AF production originate from the field or additional AF are produced during the fermentation phase or during aerobic deterioration of corn silage. The trial was carried out in northern Italy on corn at a dry matter (DM) level of 34%. The fresh herbage was either not treated (C) or treated with a Lactobacillus buchneri (LB) NCIMB 40788 [(at 3 × 10⁵ cfu/g of fresh matter (FM)], Lactobacillus hilgardii (LH) CNCM I-4785 (at 3 × 10⁵ cfu/g of FM), or their combination (LB+LH; at 1.5 × 10⁵ cfu/g of FM of each strain) ensiled in 20-L silos and opened after 250 d of ensiling. After silo opening, the aerobic stability was evaluated and samples were taken after 7 and 14 d of air exposure. The pre-ensiled material, the silages at silo opening, and the aerobically exposed silages were analyzed for DM content, fermentative profiles, microbial count, nutritive characteristics, DM losses, and AFB₁, AFB₂, AFG₁, and AFG₂ contents. Furthermore, a subsample of colonies with macromorphological features of Aspergillus section Flavi was selected for AF gene pattern characterization and in vitro AF production. The presence of A. flavus was below the detection limit (<1.00 log₁₀ cfu/g) in the fresh forage before ensiling, whereas it was found in 1 out of 16 silage samples at silo opening at a level of 1.24 log₁₀ cfu/g. The AF were found in both the fresh forage and at opening in all the samples, with a predominance of AFB₂ (mean value of 1.71 μg/kg of DM). The inoculation of lactic acid bacteria determined a reduction in the lactic-to-acetic ratio compared with the control. A larger amount of acetic acid resulted in a lower yeast count and higher aerobic stability in the treated silages than in the control ones. At the beginning of aerobic deterioration, the yeasts increased to over 5 log₁₀ cfu/g, whereas the molds were close to the value observed at silo opening. When the inhibiting conditions were depleted (pH and temperature higher than 5 and 35°C, respectively), both the total molds and A. flavus reached higher values than 8.00 and 4.00 log₁₀ cfu/g, respectively, thus determining the ex novo production of AFB₁ during aerobic deterioration, regardless of treatments. The analysis of gene pattern showed that 64% of the selected colonies of A. flavus showed the presence of all 4 AF gene patterns, and 43% of the selected colonies were able to produce AF in vitro. During air exposure, after 1,000°C·h have been cumulated, starch content decreased (below 10% DM) and concentration of neutral detergent fiber, acid detergent fiber, hemicelluloses, crude protein, and ash increased. The inoculation with LB and LB+LH increased the aerobic stability of the silages and delayed the onset of aerobic microbial degradation, which in turn indirectly reduced the risk of A. flavus outgrowth and AFB₁ production after silage opening.     

Molecular identification of cephalopod species by FINS and PCR-RFLP of a cytochrome b gene fragment

Identification of commercial species is a relevant issue to assure the correct labeling of seafood products. In this work two different molecular techniques, FINS (Forensically Informative Nucleotide Sequencing) and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) were developed to identify 8 cephalopod species (families Loliginidae and Ommastrephidae) employing a fragment of the cytochrome b gene. DNA amplification for all of the species was carried out with a new set of specific primers designed in this study for cephalopods. FINS is a technique based on DNA sequencing, while PCR-RFLP allows direct species identification by comparing specific DNA restriction patterns. Both techniques are useful for cephalopod species identification. 17 food products (mainly "squid rings") were analyzed and the species employed for their manufacture identified by FINS.     

Aspergillus Section Flavi and aflatoxins in dusts generated by agricultural processing facilities in the Philippines

Relatively few data exist regarding concentrations of aflatoxins and their causative organisms in dusts within occupational environments. Here, we examined Aspergillus Section Flavi populations and aflatoxin levels in 54 samples of dusts generated by agricultural processing facilities as possible indicators of aflatoxin exposure in the Philippines. The average incidence of Aspergillus Section Flavi expressed as a percentage of total mould populations in rice dust, corn dust, feed dust and copra dust were 8, 4, 31 and 10%, respectively. Predominant aflatoxigenic fungi isolated were Aspergillus flavus and A. parasiticus with ratios of 31:1, 40:5, 16:4 and 1:1 in rice dust, corn dust, feed dust and copra dust, respectively. Aflatoxins produced by selected isolates in in vitro rice culture ranged from 100 µg kg^?1 to 100.5 mg kg^?1. Toxigenicity of isolates based on the average aflatoxin concentrations produced by positive isolates were in the order of copra dust > corn dust > rice dust > feed dust. Average natural concentrations of aflatoxins in rice dust, corn dust, feed dust, and copra dust were 25, 6, 15 and 10 µg kg^?1, respectively. Estimates of the amount of inhaled aflatoxins by workers in an 8‐h work shift ranged from 0.06 to 114 ng, the average of which is higher than the amount of aflatoxins ingested by Filipinos due to the consumption of polished rice. The presence of highly toxigenic Aspergillus Section Flavi and aflatoxins in agricultural dust is a critical health risk for workers, considering the frequency of exposure and the possibility of inhalation and subsequent absorption of aflatoxins in the respiratory tract. Copyright © 2006 Society of Chemical Industry     

利用线粒体DNA Cyt b基因PCR-RFLP分析方法鉴别羊肉和鸭肉

建立了一种利用线粒体DNA（mtDNA） Cyt b基因PCR-RFLP分析来鉴别羊肉和鸭肉的方法。采用一对通用引物扩增绵羊、山羊和鸭的mtDNACytb基因,并对扩增产物用DNA限制性内切酶Bsu36I和SpeI进行酶切,电泳分析酶切产物的变化。结果表明通用引物可扩增羊和鸭472bp的PCR产物,经两种内切酶酶切后,绵羊、山羊和鸭的PCR产物分别被切为大小不同的片段,其中绵羊和山羊被SpeI切为213bp和259bp,而鸭则被Bsu36I切为95bp和377bp。利用PCR-RFLP分析mtDNA Cyt b基因的方法操作简单,是一种快速鉴别羊肉和鸭肉的可靠方法。      

Polymerase chain reaction (PCR) identification of Aspergillus niger and Aspergillus tubingensis based on the calmodulin gene.

Aspergillus niger and A. tubingensis, species belonging to section Nigri, are commonly found in plant products and processed food, such as grapes, cereals, coffee, and derived products. These two species are very difficult to differentiate by classical morphological criteria and some isolates are known to produce ochratoxin A. The exact identification of these two species is very important to avoid the overestimation of toxicological contamination and related risks. A polymerase chain reaction (PCR)-based identification and detection assay was developed as a tool to identify A. niger and A. tubingensis, using molecular differences obtained by sequencing the calmodulin gene. Two pairs of species-specific primers were designed and empirically evaluated for PCR identification of A. niger and A. tubingensis. Species-specific PCR products generated by each primer set were 505 bp (A. tubingensis) and 245 bp (A. niger) in length, which could be potentially useful for a multiplex PCR assay. The sensitivity of this assay was about 10 pg DNA in a 25-microl PCR reaction volume, using pure total DNA of the two species. The method described in this study represents a rapid and reliable procedure to assess the presence in food products of two ochratoxigenic species of section Nigri.     

Identification of gadoid species (Pisces, Gadidae) by sequencing and PCR–RFLP analysis of mitochondrial 12S and 16S rRNA gene fragments

We applied PCR–RFLP (Polymerase Chain Reaction – Restriction Fragment Length Polymorphism) analysis to identify seven gadoid species of different biogeographical origin and commercial relevance, namely Gadus morhua (Atlantic ocean); Trisopterus minutus capelanus, Trisopterus minutus minutus, Molva elongata, Phycis blennoides, Micromesistius poutassou (Atlantic ocean and Mediterranean sea); Theragra chalcogramma (Pacific ocean). Two DNA fragments belonging to mitochondrial 12S and 16S rRNA genes of about 430 and 630 bp, respectively, were isolated by PCR amplification. Their direct sequencing showed a significant genotypic diversity among gadoid species, useful for species identification. Digestion of 16S rRNA gene PCR fragment with MvaI or Bsh1285I restriction enzymes, followed by agarose gel electrophoresis of the cleaved products, yielded specific restriction profiles that enabled direct, visual identification of the species analyzed. This PCR–RFLP method allowed a clear and rapid discrimination of the gadoid species studied.