Development of a rapid and simple molecular identification methodology for true sardines (Sardina pilchardus) and false sardines (Sardinella aurita) based on the real-time PCR technique

Sardines are a small pelagic species belonging to the Clupleidae family and have high commercial value because of the properties of their meat. One of the most valuable fish in this family is Sardina pilchardus, which in addition to its high commercial value is the one that should be labeled on canned sardines, the other species being labeled as sardines X. In this study, a fast real-time polymerase chain reaction (PCR) assay based on TaqMan probe technology, which amplifies a fragment of the internal transcribed spacers (ITS1 and ITS2) and the 5.8 S rRNA region, was developed for the authentication of two commercially important species of sardines, S. pilchardus and S. aurita. This methodology can be applied to a variety of products, including those that have been subjected to intensive processing treatments such as canning. The present methodology was validated and subsequently applied to 50 commercial samples in order to determine whether correct labeling is being carried out in the market. This assay combines the high specificity, sensitivity, and rapidity of fast real-time PCR with the advantages of using two different probes in the same reaction, making it a powerful tool for verifying the correct application of food labeling regulations.     

Rapid method for controlling the correct labeling of products containing European squid (<Emphasis Type="Italic">Loligo vulgaris</Emphasis>) by fast real-time PCR

The squids are a group of cephalopods widely distributed and with high commercial value. In the present work, a fast real-time PCR was developed for the authentication of the European squid (Loligo vulgaris). This method is based on a specific primer/probe set that amplifies a fragment of the Internal Transcribed Spacer 1 (ITS 1) ribosomal DNA region. This technique is notable for its conceptual and practical simplicity, together with its combination of speed, sensitivity and specificity in a homogeneous assay. To all this must be added the time savings produced by the fast real-time PCR due to shorter runs. The presented methodology was validated to check how the degree of food processing affects the applicability of this technique and therefore the detection of L. vulgaris. It was demonstrated that the technique can be applied to all kinds of processed products. The commercial denomination of some cephalopods, including European squids, is an important issue due to the legal gaps, since the same species has different commercial name depending on the format in the market. The methodology herein developed was applied to 42 commercial samples to evaluate the situation regarding the labeling of products made from these species. Moreover, the method can be applied to all kinds of products regardless of the degree of processing.     

Identification of Ingredient in Mullet Roe Products by the Real-Time PCR Method

Mullet roe is a product of high economic value in a number of Asian countries, particularly Taiwan. However, actual mullet roe is commonly adulterated by the addition of other species, such as escolar and oilfish. The purpose of this study was to develop a method to detect the ingredient of mullet roe products. Based on the TaqMan real-time PCR assay, we designed the specific primers-probe set (mullet) that targets the mitochondrial 16S ribosomal RNA gene. Meanwhile, the positive amplification control is designed based on the eukaryotic 18S rRNA gene. The PCR amplicon used to identify fish species in processed roe products is smaller than 200 bp. Method specificity was evaluated by analyzing tissue samples of 29 food fish and 9 puffer fish species. No indications of cross-reactivity toward non-target species were observed. Sensitivity and linearity tests were conducted using five-fold serial dilutions of target DNA from processed mullet roe, and we determined that our proposed method has a sensitivity of 1.2 ng. Further tests on a random survey of commercial fish roe products demonstrated the efficacy of the technique in the detection of mullet DNA. The real-time PCR methods developed in this study could be used to verify the labeling of actual mullet roe products.     

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.     

Duplex real-time PCR for authentication of anglerfish species

A duplex real-time PCR assay based on TaqMan probe technology was developed for the authentication of two commercially important anglerfish species (Lophius budegassa and L. piscatorius). This technique uses the cytochrome oxidase subunit I and allows the unambiguous identification of this species. It is notable for its simplicity, rapidity, highest potential for automation and minor risk of contamination. The TaqMan real-time PCR is currently the most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of these species. The method can be applied to all kind of products as fresh, frozen, and precooked products. The developed methodology, which uses two specific primer–probe sets, has been validated and subsequently applied to 40 commercial samples labeled as any anglerfish species in order to determinate whether the species used for their manufacturing correspond to these species. The methodology herein developed is appropriate to clarify questions related with the correct labeling of commercial products, traceability in commercial trade, and fisheries control.     

Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products

Fast real-time PCR TaqMan assays were developed and validated for species identification in dairy products. Based on the amplification of 12S rRNA and cytB partial genes of mitochondrial DNA, the methods were demonstrated to be sensitive, fast, and species-specific for Bos taurus, Ovis aries, Bubalus bubalis, and Capra hircus. The limit of detection calculated was lower than 1%, and the efficiency was reported to be higher than 96% in every assay. An internal amplification control was used to detect possible false negatives. The method was validated by means of laboratory-prepared samples mixing different species. Moreover, 18 commercial dairy samples were analyzed by both real-time PCR and isoelectric focusing, the official European Union reference method. The 4 TaqMan assays were confirmed to be a useful tool for milk and dairy product authentication.     

Detection of rabbit and hare processed material in compound feeds by TaqMan real-time PCR

Food and feed traceability has become a priority for governments due to consumer demand for comprehensive and integrated safety policies. In the present work, a TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for specific detection of rabbit and hare material in animal feeds and pet foods. The technique is based on the use of three species-specific primer/probe detection systems targeting three 12S rRNA gene fragments: one from rabbit species, another one from hare species and a third fragment common to rabbit and hare (62, 102 and 75 bp length, respectively). A nuclear 18S rRNA PCR system, detecting a 77-bp amplicon, was used as positive amplification control. Assay performance and sensitivity were assessed through the analysis of a batch of laboratory-scale feeds treated at 133°C at 3 bar for 20 min to reproduce feed processing conditions dictated by European regulations. Successful detection of highly degraded rabbit and hare material was achieved at the lowest target concentration assayed (0.1%). Furthermore, the method was applied to 96 processed commercial pet food products to determine whether correct labelling had been used at the market level. The reported real-time PCR technique detected the presence of rabbit tissues in 80 of the 96 samples analysed (83.3%), indicating a possible labelling fraud in some pet foods. The real-time PCR method reported may be a useful tool for traceability purposes within the framework of feed control.     

Real-time PCR method for the detection of figwort mosaic virus (FMV) to complement the FMV 34S promoter-specific PCR assay used for screening of genetically modified plants

In this study a new real-time PCR assay for the detection of figwort mosaic virus (FMV) DNA is described. This assay targets a 113-bp-long sequence of the FMV open reading frame Vll, a non-conserved coding region among the caulimoviruses. Detection of FMV DNA is useful to complement screening for the FMV 34S promoter (P-FMV), a genetic element present in several genetically modified (GM) plants. The specificity of the assay was assessed against closely related plant viruses, plant species of agronomic importance or susceptible to infection by FMV, and various GM plants containing the P-FMV sequence. No cross-reactivity of the assay against the tested nontarget organisms was observed. The limit of detection of the FMV real-time PCR assay was determined at approximately 5 copies per reaction. The robustness of the method was tested using different real-time PCR instruments and PCR master mixes with no negative effects on the PCR efficiency and linearity being observed. When amplifying the FMV target DNA at a concentration level close to the detection limit, no negative influence on the PCR performance was observed in the presence of background genomic DNA from various plant species. The precision data of the in-house validation were in line with generally accepted performance requirements. In summary, the method appears fit for the purpose of a specific identification test for the presence of genomic FMV DNA, while preventing false-positive results during P-FMV screening.     

Identification of Flatfish Species Using Polymerase Chain Reaction (PCR) Amplification and Restriction Analysis of the Cytochrome b Gene

Restriction site analysis of PCR products from a conserved region of the cytochrome b gene has been used for the specific identification of sole ( Solea solea ), European plaice ( Pleuronectes platessa ) and flounder ( Platichthys flesus). Polymerase chain reaction (PCR) amplification of the cytochrome b gene using universal primers produced a 359 bp fragment in all species analyzed. Digestion of the PCR products with Nci I, Sau 3AI and Hinf I endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of the fish species. This methodology should prove useful for enforcing labeling regulations in the authentication of flatfish species.     

Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products

The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg^?1 for sesame and 1.4 mg kg^?1 for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.     

High resolution melting analysis for quantitative detection of bovine milk in pure water buffalo mozzarella and other buffalo dairy products

Identification of buffalo dairy products has become an important issue to ascertain product quality, consumer rights and absence of food-borne allergic reactions. A polymerase chain reaction (PCR) followed by a high resolution melting (HRM) analysis was developed and applied for species specific detection of bovine milk in nine different commercial buffalo dairy products. A specific buffalo 12S rRNA and a bovine d-loop primer pair, targeting the mitochondrial genome, were employed in a duplex PCR assay. The analysis developed was found capable of identifying the presence of bovine milk down to 1% in commercial buffalo milk products and also of quantifying the ratio of bovine into buffalo milk. HRM was proven to be a fast and accurate technique for a routine authentication testing of mozzarella and other buffalo milk products.     

Detection of Fish-Derived Ingredients in Animal Feeds by a TaqMan Real-Time PCR Assay

Animal species identification in food and feed has gained increasing interest in recent years due to public health, economic and legal concerns. In the present work, a TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for detection of a specific DNA fragment (87 bp) common to the most economically important fish families in fish-derived ingredients used in feed formulations. Performance of the assay was evaluated against stringent acceptance criteria in terms of specificity and sensitivity. The TaqMan real-time fish-specific system proved to be highly sensitive, allowing the detection of 0.1 pg of fish DNA. The assay was successfully applied to the authentication of two types of real-world compound feeds: industrial farm animal feeds and commercial pet foods, allowing detection of small fish percentages in the samples. The quantitative potential of the real-time PCR assay was further performed, concluding that, although results were well reproducible under the experimental conditions tested, an accurate estimation of the target content in feeds is not feasible in practice. The reported methodology may be useful as a molecular analytical tool to support fisheries control and enforcement, as well as the verification of authenticity of fish and fish-derived products in the food and feed sectors.     

Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.     

The detection of commercial duck species in food using a single probe-multiple species-specific primer real-time PCR assay

A real-time PCR assay for the simultaneous detection of Mallard and Muscovy duck is described. Species-specific primers were designed for Mallard or Muscovy duck using the mitochondrial cytochrome b gene sequence. These primer sets were multiplexed with a single duck probe to produce a simple, rapid and robust real-time PCR assay. This assay was shown to be specific for duck compared to a wide range of commercially important meat species and was used for the successful detection of duck meat in complex food matrices. This is the first report of an assay that will detect all species of commercially available duck in commercial products using real-time PCR.     

A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis

The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.     

Development of a quantitative real-time PCR method to enumerate total bacterial counts in ready-to-eat fruits and vegetables

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.     

实时荧光PCR法鉴定食品中双歧杆菌

根据双歧杆菌属16S rRNA基因的保守区序设计特异性引物和探针,建立一种鉴定食品中双歧杆菌属的实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法。对方法的特异性、灵敏度、重复性和体系抗干扰能力进行验证,最后采用该方法对市售25 份标示含双歧杆菌样品进行检测。结果表明：该检测方法可特异性检测双歧杆菌属细菌,对近缘的乳杆菌属、链球菌属及食品中常见菌群包括大肠杆菌、金黄色葡萄球菌、沙门氏菌等均无扩增。双歧杆菌DNA检测绝对灵敏度可达到2 pg,相对灵敏度可达到104 CFU/mL。重复性测试表明相对标准偏差小于1%。同时进行了杂菌干扰检测实验,在培养物水平和纯基因组DNA水平上将青春双歧杆菌ATCC15703与大肠杆菌ATCC25922混合进行检测,检出Ct值较纯菌检测时无显著影响,表明建立的荧光PCR方法抗干扰能力良好。对25 份市售实际样品进行测试,有5 份标识含有“双歧杆菌”的样品未检测出双歧杆菌成分。本研究所建立的实时荧光PCR法能准确、快速检测食品中双歧杆菌属细菌。     

Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.