Ahmad Nizar Nina Naquiah, Ali Md Eaqub, Hossain M A Motalib, Sultana Sharmin, Ahamad Mohammad Nasir Uddin
a Nanotechnology and Catalysis Research Centre (NANOCAT) , University of Malaya , Kuala Lumpur , Malaysia.
b Centre for Research in Biotechnology for Agriculture (CEBAR) , University of Malaya , Kuala Lumpur , Malaysia.
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2018 Jun;35(6):1038-1051. doi: 10.1080/19440049.2018.1440644. Epub 2018 Feb 26.
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.
由于鳄鱼肉具有独特的风味和感官吸引力，且胆固醇和脂质含量低，对其需求正在迅速增长。此外，自古以来，鳄鱼油和鳄鱼血就被用于替代药物中治疗哮喘和其他几种疾病。此外，鳄鱼皮在皮革工业中需求量很大。所有这些因素共同导致了在世界大部分地区对鳄鱼的广泛捕猎、非法交易以及随之而来的数量下降。为了满足不断增长的需求，一些鳄鱼品种，如湾鳄，已在养殖场养殖，其商业贸易也已合法化。然而，对野生鳄鱼在食品和药品方面的需求仍居高不下。最近，有人提出了几种基于DNA的鳄鱼检测方法，但这些检测方法基于单个基因和较长片段的扩增子靶标，在大量处理过程中会分解。为了填补这一空白，我们开发并验证了一种高度稳定的双基因靶向多重PCR检测方法，用于鉴定商业产品中的湾鳄材料。该检测方法涉及湾鳄atp6基因（77 bp）和cytb基因（127 bp）的两个短片段以及真核生物的通用内对照（99 bp）。PCR引物针对18个物种进行了交叉测试，并在广泛的煮沸、高压灭菌和微波烹饪条件下的纯基质和混合基质中进行了验证。最后，它被用于鉴定五种基于鳄鱼的商业产品。在纯肉中，atp6和cytb基因的检测下限分别为0.001 ng和0.01 ng DNA，在混合基质中为1%。一些固有特征，如77 - 127 bp的扩增子大小、出色的稳定性和卓越的灵敏度，表明该检测方法可用于鉴定任何法医样本中的湾鳄。
