RT-PCR Based Test for Species Specific Milk and Milk Products
In recent times, with increasing consumption of dairy food items, the species identification of milk and milk products has received a great attention. It has a significant importance for several reasons related to government regulations, religion and public health. The extensive consumption of milk and dairy products makes these foodstuffs targets for potential adulteration with financial gains for unscrupulous producers (Nicolaou et al., 2011). In the dairy sector, the fraudulent misdescription of food contents on product labels has been reported especially with high added value milk products commanding a premium price. The description and labeling of food must be accurate so that consumers can make informed choices about their diet and the products they buy (Herman, 2001; Herna´ndez et al., 2003) Testing authenticity of food products such as milk, meat and fish is important for labeling and assessment of value and is therefore important to prevent unfair competition and assure consumers protection against fraudulent practices commonly observed in the food industry (Xue et al., 2010). The majority of dairy products’ authenticity testing methodologies are based on major milk proteins analysis (Stanciuc and Rapeanu, 2010). Different analytical approaches have been used for milk species identification such as immunological (Xue et al., 2010; Zelenakova et al., 2008; Hurley et al., 2004), electrophoretical (Mayer, 2005) and chromatographic (Enne et al., 2005). With recent advances in DNA technology, DNA based methods are been used for many aspects of food authentication, including milk adulteration detection (Woolfe and Primrose, 2004).Molecular techniques have proved to be reliable, sensitive and fast. Among molecular techniques, PCR is the most widely used test for the identification of species of origin in milk (Bottero et al., 2003).Recent studies have shown that it is possible to use milk as a source of DNA and as a substrate for PCR. Ruminant milk from healthy mammary glands has a large amount of somatic cells which contain genomic DNA suitable for PCR amplification (Amills et al., 1997; Lipkin et al., 1993; Murphy, Reza et al., 2002). Accurate species identification by PCR is highly dependent on the specificity of primers used. These primers should target a DNA fragment with sufficient species to species variation. It is very common in India that with low priced cow milk adulterated high priced buffalo milk. Various techniques are available in market to identify the origin of milk but all these techniques have their own limitations. Thus, DNA based technologies are preferred now a days to identify the origin of food products from animal sources due to high sensitivity and specificity. Currently a PCR based method was developed to identify cow milk in raw buffalo milk. DNA was isolated from milk by DNeasy Mericon food kit (Quiagen,USA) which resulted in DNA of requisite quality for downstream applications. A bovine specific primer targeting D-loop (displacement) of mt- DNA (mitochondrial) was selected and standardized to amplify cow DNA. Specificity of primer was tested across the species in the genomic DNA isolated from both milk and blood. The protocol can be sensitive to detect upto 5% level of cow milk in the buffalo milk. Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in “Food Genomics;” a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or “end-point” PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA.
Real-time PCR
PCR (short for polymerase chain reaction) is an important analysis method in food analysis as well as in medical diagnostics.
A real-time PCR test duplicates and analyzes specific DNA sequences. In food analysis, PCR tests are used in many applications, e.g. for the detection of pathogenic microorganisms, for allergen identification, for the detection of genetically modified organisms (GMO) or for the identification of animal species. PCR tests offer many advantages: They are very specific, sensitive, robust, rapid, reliable and moreover, the tests can be automated.
- Safe: specific and validated method
- Reliable: quality-controlled test kits
- User-friendly: easy-to-use, universal sample preparation
- Effective: capable to multiplex and quantify
- Versatile: many application areas
CALF-LAB
DNA-based approaches for dairy products authentication
Compiled & Shared by- Team, LITD (Livestock Institute of Training & Development)
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Reference-On Request.
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