Results (
Thai) 2:
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two endonucleases, NlaIII and MseI, were determined to be
potentially useful for species identification. Samples I1, I7, T1, and
T2 contained restriction sites that produced 4 major fragments of
approximately 182, 50, 42, and 32 bp. These results were compared
with those in Table 2, indicating that these samples matched the Cy.
carpio carpio sequence analysis. Samples I2eI6 and I8eI10 produced
3 fragments of approximately 161, 100, and 32 bp after codigestion.
These results matched the Ct. idella sequence analysis.
Samples I11eI14 and T6eT10 contained the restriction site that
produced 4 major fragments of approximately 200, 50, 42, and
32 bp. These results matched the sequence analysis of C. auratus
auratus. To summarize the above results, the 24 Cyprinidae-related
products were identified as C. auratus auratus, Cy. carpio carpio and
Ct. idella. The results also indicated that there were no counterfeits
or frauds in this study. This finding was similar to previous reports
(Coïsson et al., 2010; Dooley, Sage, Brown, et al., 2005; Dooley, Sage,
Clarke, et al., 2005;Wang et al., 2009). The current results indicated
that the QIAxcel automated CE system provided a method of species
identification using PCR-RFLP that had high-resolution separation,
saved time, and used fewer reagents and smaller sample
sizes, suggesting that the CE method is a good application of molecular
techniques for routine food authentication analyses.
To understand the relationship between thermal processing
and DNA degradation in species identification, we exposed the
common carp species Cy. carpio carpio to different heating processing
treatments and later amplified the DNA using the CypbL/
CypbH primer set. The amplification limit is shown in Fig. 5. The
results showed that a 331-bp fragment was still amplified from
the fish meats when they were heated at 121 C for 15 and 30 min
or 160 C for 3, 5, 7, and 9 min. The DNA was seriously degraded
when the fish meat was heated at 121 C for 45 min and 160 C for
11, 13, and 15 min, and these samples could not be amplified by
CypbL/CypbH. The result was similar to that of other reports
(Hsieh, Chai, Cheng, Hsieh, & Hwang, 2004; Hsieh et al., 2010).
Hsieh et al. (2010) indicated that when the DNA fragments were
degraded, it was difficult to obtain fragments larger than 400 bp.
Hsieh et al. (2004) noted that for swordfish mtDNA, fragments of
both 137 and 87 bp were amplified when the mtDNA was
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