This research will contribute to the development of an
external standard for qPCR-based quantification and a routine inspection
protocol for instrument performance and kit and interlaboratory-user
capabilities in national-wide inspections. Authenticity and traceability are
essential for modern food and medicine inspection, and reliable techniques are
important for the trade of halal foods, which reach more than 20 percent of the
world market. Porcine ingredients can be a potential source of food
adulteration when fraudulently added to processed foods without proper
labeling. A sensitive and accurate porcine detection method is required to
develop a conformity assessment system that includes laboratory testing for
porcine-free certification. This study proposes a procedure that could be
incorporated into the development of a standardized control and protocol for
real-time PCR (qPCR) methods and their traceability using droplet digital PCR
(ddPCR). The design used a recombinant pUC57 plasmid as an amplification target
to carry the 97 bp fragment of the porcine ATCB gene. The absolute
quantification and linearity assessment showed high precision with R2 values of
0.9971 and 0.9998 for qPCR and ddPCR, respectively. In general, both methods
showed comparable results in terms of linearity and detection limit. Beta-actin
and other single-copy genes are less effective than mitochondrial DNA (mtDNA)
for assessing the authentication of porcine testing, possibly because mtDNA is
abundant in cells, thereby increasing its amplification probability. However,
both limits of detection assessments showed high sensitivity, although ddPCR
showed a slightly higher sensitivity than that of qPCR, especially at low DNA
concentrations. Evaluations of multiple-sample and inter-participatory testing
demonstrated the qPCR method's excellent sensitivity, wide application, and
robustness. Consequently, we draw the conclusion that the digital PCR approach
yielded more reliable results based on a low quantity (less than five copy
number) recombinant plasmid study. These findings may offer scientific data
that regulatory bodies, particularly those in Indonesia, can use to build and
formulate a reliable qPCR procedure for porcine detection that makes use of
predicted DNA quantities.
Author(s) Details
Umi Nuraeni
Laboratory of National Measurement Standards of Biology, The
National Standard Agency of Indonesia (BSN), South Tangerang, Banten,
Indonesia.
Retno Tri Astuti
Department of Fisheries Product Technology, Faculty of Fisheries
and Marine Science, Universitas Brawijaya, East Java, Indonesia.
Jekmal Malau
Department of Pharmacy, Faculty of Health Science,
Universitas Singaperbangsa Karawang, West Java, Indonesia.
Auraga Dewantoro
Research Center for Genetic Engineering, The National
Research and Innovation Agency of Indonesia (BRIN), Bogor, Indonesia.
Dini Apriori
Laboratory of National Measurement Standards of Biology, The
National Standard Agency of Indonesia (BSN), South Tangerang, Banten,
Indonesia.
Evellin Dewi Lusiana
Department of Aquatic Resource Management, Faculty of
Fisheries and Marine Science, Universitas Brawijaya, East Java, Indonesia.
Bambang Prasetya
Research Center for Testing Technology and Standards, The
National Research and Innovation Agency of Indonesia (BRIN), South Tangerang,
Indonesia.
Please see the link:- https://doi.org/10.9734/bpi/strufp/v11/1654
