Establishment Of Loop-mediated Isothermal Amplification For Visible Detection Of Animal Ingredients

Meat and its products play important roles as sources of protein in our daily life.However, the meat product authenticity problem had been reported several times inChina in recent years. Disclosing the constituents of meat products to consumers isnecessary. Although there are many countries, such as Malaysia, Indonesia andCanada, that have clear and definite regulations stating that halal food compositionmust be labeled in detail, food adulteration is still an international problem. Moreover,the regulation about food security in China still need to be improved. Therefore, it isnecessary to develop a reliable, sensitive and rapid method to identify undeclaredcomponents in raw, adulterated and processed meat products.Several analytical methodologies have been developed, including fouriertransform infrared spectroscopy, liquid chromatography with electrochemicaldetection, enzyme-linked immunosorbent assay, the polymerase chain reaction (PCR),species-specific PCR, PCR-restriction fragment length polymorphism (PCR-RFLP),SYBR green real-time PCR and TaqMan probe real-time PCR. Some of theseanalytical approaches have been used in various countries and organizations asdetection method for commercial meat products. However, these methods rely onlaboratory and operating of special equipment, which limit the usage of thesemethods.Loop-mediated isothermal amplification (LAMP) is a new nucleic acidamplification method. This method relies on four specific primers, inner primers (FIPand BIP) and outer primers (F3and B3), that recognize six distinct regions of thetarget DNA and have high sensitivity and specificity. In addition, the LAMP methodhas the significant advantages of rapidity and ease of operating. Upon incubation in asimple incubator, DNA is amplified under isothermal conditions (60-65℃) inapproximately60min by the Bst DNA polymerase. Foremost, during theamplification of the DNA, a large number of pyrophosphate ions is produced, and the mixture thus exhibits visible color in the presence of calcein. Therefore, due to thecolor-based character of the LAMP assay, this method can be utilized in on-the-spotdetection. Recently, LAMP detection methods have been widely used for transgenicplants, microorganisms and viruses. However, only a few methods have been reportedfor meat product identification.In this study, we developed a rapid method based on loop-mediated isothermalamplification for visibly identify pork, fox, rat, chicken, and duck DNA in meatproducts, respectively. Specific primers were designed according to porcine ND1gene,fox cytb gene, rat and chicken D-loop region and duck COX3gene sequence,respectively. The resuls demonstrated that each pair of primers can specificallyidentify differnent meat ingredients. The sensitivity of the LAMP assay for pork, fox,rat, chicken and duck DNA detection were500fg,500fg,50fg,5pg and50fg,respectively. The0.01%adulterated sample can be detected specifically, regardlessthe processing temperature. Furthermore, the LAMP assay was compared with theindustry standard methods of Chinese entry-exit inspections and quarantines (SN/T2051-2008). The result showed that the consistent rate between these methods was97.6%, indicating the assay established in this study can be used in rapid detection ofmeat product identification.In conclusion, the LAMP assay established in this study has excellent sensitivityand specificity and is a convenient method for meat product identification.