基于行业标准中马和驴成分检测方法用于骡肉检测的分析

Application of Industrial Standard Methods for Detection of Horse-and Donkey-Derived Ingredients for Detecting Mule Meat

应用基于线粒体基因的行业标准检测方法SN/T 3730.4—2013《食品及饲料中常见畜类品种的鉴定方法第4部分:驴成分检测实时荧光PCR法》和SN/T 3730.5—2013《食品及饲料中常见畜类品种的鉴定方法 第5部分:马成分检测实时荧光PCR法》检测骡肉时,存在马、驴成分均可检出的情况,与线粒体严格母系遗传的理论相左。因此,本研究通过测序法和行业标准中的马、驴成分检测方法对骡肉进行检测,对SN/T 3730.4—2013用于骡肉检测的结果进行分析和讨论。本研究基于线粒体基因和核基因聚合酶链式反应(polymerase chain reaction,PCR)测序、SN/T 3730.4—2013、SN/T 3730.5—2013方法对9份单一肉块样品进行了检测。结合线粒体基因和核基因的检测结果,确定3份骡肉均为马骡肉;采用SN/T 3730.4—2013和SN/T 3730.5—2013方法对3份马骡肉均能检出驴成分和马成分,SN/T 3730.5—2013对马骡肉中马成分检测的循环阈值(cycle threshold,Ct)≤20.00;SN/T3730.4—2013对马骡肉中驴成分检测的Ct值在25.00~35.00范围内,且以SN/T 3730.4—2013中引物进行的普通PCR产物测序结果显示,3份骡肉与马、驴均不同源。分析认为,出现该现象的原因可能是SN/T 3730.4—2013靶序列以核内线粒体DNA片段形式和较低的重复数出现在马骡核基因组中,并且发生了部分碱基的插入、缺失。采用行业标准SN/T 3730.4—2013和SN/T 3730.5—2013对已知来源于单一动物源性成分的样品进行马、驴成分检测时,当马成分检测的Ct值≤20.00,同时驴成分检测的Ct值在25.00~35.00范围内时,应考虑马骡成分存在的可能性。

Both horse-and donkey-derived ingredients have been detected in mule meat by real-time polymerase chain reaction(PCR)as described in China’s industry standards for detection of horse(SN/T 3730.5-2013)and donkey(SN/T 3730.4-2013)ingredients in food and feed,respectively.This contradicts the theory of strict maternal inheritance of mitochondrial DNA(mtDNA).Therefore,in this study,3 horse meat samples,3 donkey meat samples and 3 mule meat samples were detected by mitochondrial gene and nuclear gene sequencing based on PCR and the China’s industry standard methods for horse and donkey ingredients,respectively,and the results of the SN/T 3730.4-2013 method for mule meat were analyzed.According to the results of mitochondrial gene and nuclear gene sequencing,all 3 mule meat samples were derived from mules.Both donkey and horse ingredients were detected in the 3 mule meat samples by the SN/T 3730.4-2013 and SN/T 3730.5-2013 methods.The cycle threshold(Ct)of the SN/T 3730.5-2013 method for horse ingredient was in the range of≤20.00,and that of the SN/T 3730.4-2013 method for donkey ingredient were in the range of 25.00-35.00.The sequencing results of PCR products using the primers described in the SN/T 3730.4-2013 method showed that the 3 mule meat samples had no homology with horse or donkey meat.This might be because the SN/T 3730.4-2013 target sequence appeared in the form of nuclear mitochondrial DNA segments in low repeat numbers in the mule nuclear genome,and some base insertions and deletions occurred.The possibility that mule ingredient may be present should be considered when the Ct value of the SN/T 3730.4-2013 is≤20.00,while the Ct value of the SN/T 3730.5-2013 is in the range of 25.00-35.00 for horse and donkey ingredients in known samples of single animal-derived ingredients,respectively.