Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25–153.00%, PCR efficiency of 99–100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50–7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32–28.90 ± 0.42) and melting curves (74.63– 78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth

Development and evaluation of multiplex pcr assay for the determination of cat, rabbit, rat and squirrel elements in food products / Mohammad Nasir Uddin Ahamad

Cat, rabbit, rat and squirrels are adulterated in meat and meat products for economic gain and exotic taste. However, most of these species are potential carriers of zoonotic threats and so pose huge threats to public health. Currently, several polymerase chain reaction (PCR) based methods have been proposed for authentication these species in separate assays which are costly and involved long-amplicon length biomarkers that breakdown during food processing. To overcome the need, for the first time, multiplex conventional PCR, PCR-RFLP and quantitative PCR assays with TaqMan Probes were developed here for the discriminatory identification of cat, rabbit, rat, and squirrel in food products. In conventional PCR and PCR-RFLP, rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro oven cooking. The lower limits of detection were 0.001ng DNA for pure meat and 0.1% for meatballs. Specificity was confirmed through sequencing and RFLP analysis. When PCR products were digested with BtsIMutI and BtsCI enzymes, distinctive fingerprints (115 & 8 bp for rabbit; 64 & 44 bp for rat and 176 & 67 bp for squirrel) were obtained. The detection limit of the assay was 0.1% meat in frankfurter formulation. Finally, a novel pentaplex real- time PCR assay with TaqMan probes was developed for identification and quantification of the squirrel, rat, rabbit and cat species in a single assay platform. For real-time quantitative PCR, species specific primers and probes were developed against ATP6, and cytochrome b genes to amplify 108, 123, 161and 176 bp DNA fragments from rat, rabbit, squirrel and cat meat products, respectivly under various states. A141 bp internal amplification control (IAC) of 18S rRNA was used to avoid any false negative results. Specificity of the assay was evaluated against 22 non- target species but no cross-reactivity was found. Each of the target species DNA was quantified, and PCR efficiency was determined based on standard curve that was generated using 10-fold serially diluted mixed DNA extract (1:1:1:1) from squirrel, rat, rabbit and cat species. The assay was valid both under pure, processed and admixed states with 10-0.1% (w/w) adulterant from each species. The limit of quantification was 0.003 ng DNA from each species. Analyses of 18 model burgers (9 chicken and 9 beef) and 18 frankfurters (9 chicken and 9 beef) revealed 91 - 122% target recovery at 0.1 - 10% adulteration. Finally, 72 commercial burgers (36 chicken and 36 beef) and 72 frankfurters (36 chicken and 36 beef) were screened but no target species was detected except IAC. Although, the study was validated using different food matrices, the shorter-aspects of amplicon length, exceptional stability under veracious treatment conditions convinced that the developed methods could be a useful tool in the identification and quantification of cat, rabbit, rat and squirrel species in any food matrices

Multiplex polymerase chain reaction-restriction fragment length polymorphism assay discriminates of rabbit, rat and squirrel meat in frankfurter products

The demands for rabbit meat are rapidly growing and Rabbitry is becoming a mean of livelihood for many youths. Rats and squirrels are very close relatives of rabbits, could be hunted freely or raised in domestic farming and so could be substituted in expensive rabbit meat. This study, for the first time, developed and validated a tetraplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify and discriminate rabbit, rat and squirrel meat under raw and processed foods. Four sets of primes amplified 123, 108, 243, and 141 bp fragments from rabbit, rat, squirrel and all eukaryotes, respectively. Specificity was confirmed through sequencing and RFLP analysis. When PCR products were digested with BtsIMutI and BtsCI enzymes, distinctive fingerprints (115 & 8 bp for rabbit; 64 & 44 bp for rat and 176 & 67 bp for squirrel) were obtained. The detection limit of the assay was 0.1% meat in frankfurter formulation

Double gene targeting PCR assay for the detection of Crocodylus porosus in commercial products

The demand for crocodile meat is quickly growing because of its exotic and organoleptic appeal and also the low content of cholesterol and lipids. Moreover, crocodile oil and blood have been used in alternative medicines for treating asthma and several other ailments since ancient times. Furthermore, crocodile hides have great demand in leather industries. All of these have collectively contributed to the extensive hunting, illegal trading and consequent decline of crocodiles in most parts of the world. To keep space with the growing demands, some crocodile species such as Crocodylus porosus have been raised in farms and its commercial trades have been legalised. However, demand for wild crocodiles in foods and medicines has continued in high gear. Recently, several DNA-based methods have been proposed for crocodile detection, but those assays are based on single gene and longer-sized amplicon targets that break down during extensive processing. To address this gap, here we developed and validated a highly stable double gene targeted multiplex PCR assay for the identification of C. porosus materials in commercial products. The assay involved two short sites from C. porosus atp6 (77 bp) and cytb (127 bp) genes and a universal internal control (99 bp) for eukaryotes. The PCR primers were cross-tested against 18 species and validated under pure and mixed matrices under extensive boiling, autoclaving and microwave cooking conditions. Finally, it was used to identify five crocodile-based commercial products. The lower limits of detection for atp6 and cytb genes were 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% under mixed matrices. Some inherent features, such as 77–127 bp amplicon sizes, exceptional stability and superior sensitivity, suggested the assay could be used for the identification of C. porosus in any forensic specimen

Tetraplex real-time PCR with TaqMan probes for discriminatory detection of cat, rabbit, rat and squirrel DNA in food products

Cat, rabbit, rat, and squirrel species are very sensitive in food products because most of them are potential carriers of zoonotic diseases and rejected in most religions and cultures. Since cats and rats are abundant in most parts of the world and their meats do not carry any value in legal markets, these meats could be considered as potential adulterants in halal, kosher, and other food markets. Rabbit and squirrel meats are also susceptible to adulteration. Therefore, both health and economic interests in rat, rabbit, cat and squirrel species are significant. In this work, a novel tetraplex real-time PCR assay with TaqMan probes was described to discriminate and identify all four species (cat, rabbit, rat, and squirrel) in a single assay platform. Species-specific primers and probes were developed against ATP6, and cytochrome b genes to amplify 108, 123, 161 and 176 bp DNA fragments from rat, rabbit, squirrel and cat meat products under various states. A 141-bp internal amplification control (IAC) of 18S rRNA was used to avoid any false-negative results. Specificity was evaluated against 22 species but no cross-reactivity was found. Efficiency of PCR assay as well as target quantification were determined based on a standard curve that was generated using tenfold serially diluted mixed DNA extract (1:1:1:1) from squirrel, rat, rabbit and cat species. The assay was valid under pure, processed and admixed states with 10–0.1% (w/w) adulterant from each species. The limit of detection was 0.1% under admixed samples and 0.003 ng DNA under pure states from each species. Analyses of 18 model burgers (9 chicken and 9 beef) and 18 frankfurters (9 chicken and 9 beef) revealed 91–122% target recovery at 0.1–10% adulteration. Finally, 72 commercial burgers (36 chicken and 36 beef) and 72 frankfurters (36 chicken and 36 beef) were screened but no target species was detected except IAC. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature

Double gene targeting PCR assay for the detection of <i>Crocodylus porosus</i> in commercial products

<p>The demand for crocodile meat is quickly growing because of its exotic and organoleptic appeal and also the low content of cholesterol and lipids. Moreover, crocodile oil and blood have been used in alternative medicines for treating asthma and several other ailments since ancient times. Furthermore, crocodile hides have great demand in leather industries. All of these have collectively contributed to the extensive hunting, illegal trading and consequent decline of crocodiles in most parts of the world. To keep space with the growing demands, some crocodile species such as <i>Crocodylus porosus</i> have been raised in farms and its commercial trades have been legalised. However, demand for wild crocodiles in foods and medicines has continued in high gear. Recently, several DNA-based methods have been proposed for crocodile detection, but those assays are based on single gene and longer-sized amplicon targets that break down during extensive processing. To address this gap, here we developed and validated a highly stable double gene targeted multiplex PCR assay for the identification of <i>C. porosus</i> materials in commercial products. The assay involved two short sites from <i>C. porosus</i> atp6 (77 bp) and cytb (127 bp) genes and a universal internal control (99 bp) for eukaryotes. The PCR primers were cross-tested against 18 species and validated under pure and mixed matrices under extensive boiling, autoclaving and microwave cooking conditions. Finally, it was used to identify five crocodile-based commercial products. The lower limits of detection for atp6 and cytb genes were 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% under mixed matrices. Some inherent features, such as 77–127 bp amplicon sizes, exceptional stability and superior sensitivity, suggested the assay could be used for the identification of <i>C. porosus</i> in any forensic specimen.</p

Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain

<p>Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from <i>ATP6</i> and <i>cytb</i> genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.</p