Zhiying Wang1, Tingting Li1, Wenjie Yu1, Lu Qiao1, Rui Liu1, Shanshan Li2,3, Yan Zhao1, Shuming Yang1, Ailiang Chen1. 1. Institute of Quality Standard & Testing Technology for Agro-Products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing, China. 2. National Key laboratory of Reliability and Electrical Equipment, School of Mechanical Engineering, Hebei University of Technology, Tianjin, China. 3. National Key laboratory of Reliability and Electrical Equipment, Tianjin, China.
Abstract
BACKGROUND: Compared with the traditional qualitative polymerase chain reaction (PCR), which only identifies the category of species, the quantitative PCR method provides a value, which is very important for appropriate penalty enforcement according to the extent of adulteration. However, most of the current quantitative PCR methods are based on mitochondrial genes, expressing different copy numbers in different cells and reducing the accuracy of quantitative results. In this study, single-copy nuclear housekeeping genes, instead of multicopy mitochondrial genes, were selected as both camel species-specific and reference genes to develop a novel normalized PCR system. RESULTS: This system had an excellent linear correlation (R2 = 0.9614) between camel milk content and Ct ratio (specific/reference genes), and allowed quantitative determination of the content of camel milk in adulterated milk samples. The accuracy was effectively validated using simulated adulterated samples with recoveries ranging from 90% to 120% and coefficient of variation less than 10%, exhibiting sufficient parameters of trueness and precision. CONCLUSIONS: The normalized PCR system based on single-copy nuclear genes is a simple, rapid and reliable method for the determination of the content of camel milk in adulterated milk samples, and also provides technical support for appropriate penalty enforcement.
BACKGROUND: Compared with the traditional qualitative polymerase chain reaction (PCR), which only identifies the category of species, the quantitative PCR method provides a value, which is very important for appropriate penalty enforcement according to the extent of adulteration. However, most of the current quantitative PCR methods are based on mitochondrial genes, expressing different copy numbers in different cells and reducing the accuracy of quantitative results. In this study, single-copy nuclear housekeeping genes, instead of multicopy mitochondrial genes, were selected as both camel species-specific and reference genes to develop a novel normalized PCR system. RESULTS: This system had an excellent linear correlation (R2 = 0.9614) between camel milk content and Ct ratio (specific/reference genes), and allowed quantitative determination of the content of camel milk in adulterated milk samples. The accuracy was effectively validated using simulated adulterated samples with recoveries ranging from 90% to 120% and coefficient of variation less than 10%, exhibiting sufficient parameters of trueness and precision. CONCLUSIONS: The normalized PCR system based on single-copy nuclear genes is a simple, rapid and reliable method for the determination of the content of camel milk in adulterated milk samples, and also provides technical support for appropriate penalty enforcement.
Authors: David K Bwambok; Noureen Siraj; Samantha Macchi; Nathaniel E Larm; Gary A Baker; Rocío L Pérez; Caitlan E Ayala; Charuksha Walgama; David Pollard; Jason D Rodriguez; Souvik Banerjee; Brianda Elzey; Isiah M Warner; Sayo O Fakayode Journal: Sensors (Basel) Date: 2020-12-07 Impact factor: 3.576