Influence of Sample Storage on the Quality of DNA Extracted from Milk of Goats Fed Conventional or Transgenic Soybean - Veterinary Science - page 200

33.1 Introduction

The use of PCR is gaining large acceptance as a reliable diagnostic approach for food
safety and traceability (Mafra et al. 2008 ), as also demonstrated by several studies on
detection of plant DNA in animal organisms (EFSA 2008 ). Fragments of nonspecific,
high-copy number gene and specific maize and soybean single copy genes (Einspanier
et al. 2001 ; Phipps et al. 2003 ;Pomsetal. 2003 ; Rizzi et al. 2008 ; Tudisco et al. 2010 )
as well as transgenic sequences (Tudisco et al. 2010 ) were detected in milk. Several
potential PCR inhibitors (fat, protein, enzyme, etc.) have been described in milk,
which can interfere with the quality of extracted DNA (Rossen et al. 1992 ). In the
present trial, we evaluated the influence of sample storage on quality, purity, and
amplification of feed DNA from milk of goats fed conventional or GM soybean.

33.2 Materials and Methods

The trial was carried out on a farm in Casaletto Spartano (province of Salerno)
where 20 pluriparous, grazing goats were equally divided into two homogeneous
groups (A and B) based on number of calving and previous milk yield. Animals
were fed concentrate (18% CP; 1.03 UFL/kg, as fed) containing conventional
(group A) or GM (group B) soybean s.e. meal. GM soybean (Roundup Ready ® ,
RR) has been modified with the EPSPS gene (encoding 5-enolpyruvilshikimate-3-
phosphate synthase protein) from the CP4 strain of Agrobacterium tumefaciens ,
which confers tolerance to the glyphosate family of herbicides. Twenty days after
delivery and every 15 days, five individual milk samples (100 ml) were collected,
immediately transported into the laboratory by fridge bag, and incubated overnight
at 4 C. The day after, each sample was skimmed and divided into six aliquots
(15 ml each) that were centrifuged for 20 min at 2,000

g at 4 C to obtain a pellet
of somatic cells. This pellet was twice washed with PBS (1 , pH 7.2, with 0.5 mM

EDTA). DNA from three pellets was immediately extracted according to the Wizard
method (Promega); concentration and quality were determined by spectrophotometry
at 260 and 280 nm (BioPhotometer, Eppendorf). The other three pellets were stored at

20 C and subsequently analyzed. The amplification and quality of DNA (three
replicates per aliquot) were assessed by endpoint PCR (Gene Amp PCR System
2400, Applied Biosystems) using the Cap144/469 primers (Bottero et al. 2003 )to
amplify a conserved portion (326 bp) of caprine mtDNA, which encodes the 12S
ribosomal RNA (12S rRNA) gene of mitochondrial DNA. In milk samples and in
conventional and transgenic soybean s.e. meal, as plant control, a fragment of the
high-copy number chloroplast gene ( trnL , 100 bp) was searched (Terzi et al. 2004 ).
Finally, species-specific primers for conventional and GM soybean were used: Le1 5/
3, which amplifies the soybean lectin gene (Kuribara et al. 2002 ); 35S 1/2 and CP4
EPSPS 1/2, which amplify, respectively, part of the 35S promoter (Lipp et al. 1999 )
derived from the cauliflower mosaic virus and part of the specific gene sequence