Use of quantitative real-time PCR to estimate maize endogenous DNA degradation after cooking and extrusion or in food products.
Polymerase chain reaction (PCR) is being used increasingly to detect DNA sequences for food quality testing for GM content, microbial contamination, and ingredient content. However, food processing often results in DNA degradation and therefore may affect the suitability of PCR or even DNA sequence detection for food quality assurance. This paper describes a novel approach using quantitative real-time PCR (qPCR) to estimate the extent of DNA degradation. With use of two maize endogenous nuclear sequences, sets of four qPCR assays were developed to amplify target sequences ranging from<100 bp to approximately 1000 bp. The maize nuclear sequences used encode chloroplastic glyceraldehyde-3-phosphate dehydrogenase and cell wall invertase. The utility of the qPCR approach for quantifying the effective concentration of maize DNA that is needed to amplify variable length DNA sequences was demonstrated using samples of maize cornmeal cooked in water for variable times, extrusion products developed using different barrel temperature and torque settings, and a range of food products from supermarket shelves. Results showed that maize DNA was substantially degraded by a number of processing procedures, including cooking for 5 min or more, extrusion at high temperatures and/or high torque settings, and in most processed foods from supermarket shelves. Processing also reduced the effective concentration of DNA sequences capable of directing amplification of the <100 bp assays as well, particularly after popping of popping corn or extrusion at a combination of high temperature and torque settings. The approach for quantifying DNA degradation described in this paper may also be of use in disciplines where understanding the extent of DNA degradation is important, such as in environmental, forensic, or historical samples.
Murray
Sarah R
SR
New Zealand Institute for Crop & Food Research Limited, P.O. Box 4704, Christchurch, New Zealand. MurrayS@crop.cri.nz
Butler
Ruth C
RC
Hardacre
Allan K
AK
Timmerman-Vaughan
Gail M
GM
D001483:Base Sequence; D018744:DNA, Plant / Q000032:analysis* / Q000737:chemistry; D005511:Food Handling / Q000379:methods*; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000235:genetics*; D003313:Zea mays / Q000235:genetics*
D018744:DNA, Plant