National Research Centre on Meat, Chengicherla, PB No 19, Uppal Post, Hyderabad, 500039 Andhra Pradesh India.
Project Directorate on Poultry, Rajendranagar, Hyderabad, 500 030 Andhra Pradesh India.
J Food Sci Technol. 2013 Feb;50(1):141-6. doi: 10.1007/s13197-011-0230-6. Epub 2011 Jan 20.
Buffalo (Bubalus bubalis) meat is a major item of export from India but export of beef i.e. meat from cattle (Bos indicus) is prohibited. Also, adulteration of buffalo meat with that of beef (meat from cattle) is a common fraudulent practice because of prohibition on cow slaughter in most states of India. Food analysts require precise identification techniques to implement such regulations. In the present study, a method of DNA extraction by Alkaline lysis from meat samples and speciation of buffalo meat using species specific Polymerase Chain Reaction (PCR) has been reported. Alkaline lysis technique is a rapid method which involves triturating meat with four volumes of 0.2N NaOH, dilution of resultant liquid extract with eight volumes of 0.2N NaOH, heating the mix 75 °C for 20 min followed by neutralization with eight volumes of 0.04N Tris HCl. Entire procedure of DNA extraction takes less than 30 min and it is economical as it involves less expensive chemicals. Method was successfully applied in animal byproducts also viz., liver, heart and kidney. For authentication of buffalo meat, pair of primers was designed based on mitochondrial D loop gene nucleotide sequence. PCR amplification using the designed primers gave amplicon of size 482 bp in buffalo and no amplification was detected in closely related species viz., cattle, sheep and goat meat samples. Results of the assay were highly repetitive and reliable. An export sample referred by export regulation authorities was also analyzed by using the Alkaline lysis method of DNA extraction and species specific PCR which enabled authentication of meat within 5 h.
水牛肉是印度主要的出口肉类产品，但牛肉（即牛的肉）出口是被禁止的。此外，由于印度大多数邦禁止屠杀牛，因此水牛肉经常与牛肉（即牛的肉）掺假，这是一种常见的欺诈行为。食品分析师需要精确的识别技术来执行这些法规。在本研究中，报告了一种从肉类样品中通过碱性裂解提取 DNA 并使用种特异性聚合酶链反应 (PCR) 对水牛肉进行分类的方法。碱性裂解技术是一种快速方法，涉及用四倍体积的 0.2N NaOH 研磨肉，用八倍体积的 0.2N NaOH 稀释所得液体提取物，将混合物在 75°C 下加热 20 分钟，然后用八倍体积的 0.04N Tris HCl 中和。整个 DNA 提取过程不到 30 分钟，而且由于涉及的化学物质较便宜，因此经济实惠。该方法还成功应用于动物副产品，如肝脏、心脏和肾脏。为了验证水牛肉的真实性，根据线粒体 D 环基因核苷酸序列设计了一对引物。使用设计的引物进行 PCR 扩增，在水牛中得到 482 bp 的扩增子，而在亲缘关系较近的物种（如牛、绵羊和山羊的肉样）中则没有检测到扩增。该检测方法的结果高度重复且可靠。出口监管机构提交的一个出口样本也通过使用碱性裂解法提取 DNA 和种特异性 PCR 进行了分析，这使得在 5 小时内能够对肉类进行认证。
