Food Research And Development 2011 9 32 9 69 Ara h1 * 518060 Ara h1 GenBank Ara h1 DNA AF432231 2 SYBR Green -CT 2 8 Ara h1 SYBR Green 3 10 2 3 10 8 copies R 2 0.993 5 3 10 3 copies 2 8 2 Ara h1 Ara h1 2.5 1.36 %<5 % 3 3 0.0~0.4 % 3 3 Table 3 Precision test A Development of Two Assays to Detect Peanut Allergen Ara h1 DNA Residue in Food Products 1 0.077 Table 4 2 0.078 3 0.078 RSD/% 0.078 0.4 % 2.6 % 5 ml50 ml 0.1 ml 0.200 0 g/l 4 CHEN Jia-jieWANG Hai-yanLiang Qiu-ni JI Kun-meiLIU Zhi-gang * 2007 1985 * 4 Determination results of recovery /mg/l /mg/l /mg/l /% /% RSD/% 1 2 3 4 5 2.188 2.232 2.166 2.254 2.232 99 101 98 102 101 100.20 1.36 70 % 1 20g/mL 15 min [1]. [J].,2006(1): 49-50 [2]. [J].,1993,24 (10):548-549 [3]. [J ].,2004(5): 67-69 [4]. [J]. 2007(3) 99-102 100.20 2010-12-24
70 Ara h1 State Key Laboratory of Respiratory Disease for Allergy at Shengzhen UniversityShenzhen UniversityShenzhen 518060GuangdongChina AbstractTo develop new methods for detection of the peanut major allergen component Ara h1 DNA in foods by using Nested and SYBR GreenReal-time quantitative technology. A DNA fragment of the peanut major allergen Ara h1 gene was amplified through the design of two pair specific primers according to data of GenBank No.AF432231. The two pairs of primers were used in Nested. The inner pair of primers for amplification of short DNA fragment was used in SYBR GreenReal-time in which the standard curve is made according to relationship between the copies and Ct value. These two methods could be used to detect peanut allergen Ara h1 DNA residue in foods. These two methods were very specific and sensitive and in SYBR GreenReal-time the linear correlation ranged from 3 10 2 to 3 10 8 copies with 0.993 5 of R 2 value and 3 10 3 copies of detection limit. Through these two assays the results of eight foods were consistent with the peanut allergen labels from their manufactures. The two methods were suitable for detection of the peanut major allergen Ara h1 DNA residue in foods with good sensitivity and accuracy Key wordsnested Real-time quantitative peanut major allergenara h1 1.1.2 [1] SYBR GreenPremix Ex Taq TM Ex Taq dntps 10 Ex Taq Buffer pmd18 -T T4 DNA [2-4] BamHI HindIII DL2000 DNA MarkerTakara DNA Omega CTAB [5-6] 1.1.3 GenBank No.AF432231 1 2 1 1.2 2 1.2.1 DNA DNA CTAB [8] [7] Real-time 8 DNA DNA OD 260 /OD 280 2 DNA Ara h 1 DNA 1.2.2 Ara h1 Ara h1 OF1Ara h1 OR1 DNA 1 1.1 DNA Ara h1 IF2Ara h1 IR2 DNA 1.1.1 DNA 25 μl DNA 1 μl Buffer 2.5 μl d NTPs 200 μmol/l Ex Taq DNA 1 U 0.5 μmol/l 25 μl 95 10 min
Ara h1 1 Table 1 Primers sequence and their usage 71 (5'to 3') 5'-GTTATCCAGCAAG Ara h1a Ara h1 OF1 GTATCAAATC-3' 5'-TAATGACTATTTGA Ara h1 OR1 TAACTACA-3' 5'-GCAGGGCAAGCCA Ara h1b Ara h1 IF2 CCGTGAC-3' 5'-GCCTCCTAACAT Ara h1 IR1 TCCTCTTGA-3' 799 bp~1 144 bp 879 bp~1 105 bp 346 bp 228 bp SYBR Green I 95 40 s 57 45 s 72 1 min 35 72 10 min 1.2.3 pmd-ara h1at/a 1.2.5.2 1 pmd18-t 3 10 10 3 10 9 3 10 8 3 10 7 3 10 6 3 pmd-ara h1a E.coli JM 10 5 3 10 4 3 10 3 1.5 10 2 copies/μl 109 LB Ct BamHIHindIII 1.2.5.3 Ara h1 DNA DNA DNA CTAB [8] 8 DNA 50 ng/μl 1.2.4 SYBR Green 1 Ara h1 IF2Ara h1 IR2 20 μl SYBR Green 2 Premix Ex Taq Buffer 10 μl 0.5 μmol/l2.1 DNA 0.5 μmol/l DNA 1 μl DNA 20 μl DNA ABI 50 ng/μl OD 260 /OD 280 1.703 DNA Prism 7300 Applied Biosystems, Warrington, UK 95 10 min, 40 95 30 s, 57 1 min 1 CTAB 8 [9] Vaǐtilingom M DNA DNA 85 ng/μl ~250 ng/μl, OD 260 / 3 10 10 3 10 9 3 10 8 OD 280 1.40~1.65 3 10 7 3 10 6 3 10 5 3 10 4 3 10 3 1.5 10 2 copies/μl2.2 Ara h1 2 ( 2 DNA) Ct Ct Ara h1 DNA 100 ng/μl DNA Ara h1 DNA DNA 50 ng/μl~200 ng/μl OD 260 /OD 280 1.48~1.75 DNA Ara h1 DNA 1 1.2.5 SYBR Green 2 1.2.5.1 Ara h1 DNA ABI Prism 7300 2 Ara h1 DNA 1 2
72 Ara h1 3 DNA 4 GenBank pmd-ara h1a 10 000 bp 7 000 bp 4 000 bp 2 000 bp 1 000 bp 500 bp M DNA Maker DL15000 1 2 3 4 5 1 DNA Fig.1 Analysis of the genomic DNA of various foods 2 000 bp 1 000 bp 750 bp 500 bp 250 bp 100 bp 2 1 DNA 1 350 bp 2 2 250 bp 2 DNA M DNA Maker 1 2 3 3 BamH Hind Fig.3 Restriction analysis for standard template vector plasmids with BamHand Hind 8 Ara h1 DNA 2 2 000 bp 1 000 bp 750 bp 500 bp 250 bp 100 bp 4 SYBR Green DNA Fig.4 DNA sequence of standard template in real-time M DNA Maker DL2000 1 2 2 3 4 1 2 DNA Fig.2 Analysis of -products of the genomic DNA from peanut by nested 2.4 SYBR Green 2.4.1 DNA 3 10 10 3 10 2 2 CT CT 2.3 pmd-ara h1a 1 pmd18-t 5 CT DNA BamH/Hind CT 350 bp CT
Ara h1 73 5 pmd-ara h1a DNA Fig.5 Amplification curve of real time with series of dilutions of standard template pmd-ara h1a 3 10 10 3 10 9 3 10 8 3 10 7 3 10 6 3 10 5 3 10 4 3 10 3 3 10 2 copies CT 3 10 2 3 10 8 copies R 2 0.993 5 6 Derivative Ct Fig.6 6 Establishment of standard curve for detection of Ara h1 DNA by real-time Table 2 7 Fig.7 Dissociation Curve 3 10 3 copies 2.4.4 DNA CTAB DNA 2.4.2 8 Ara h1 DNA 7 8 2.4.3 2 2 2 Two kinds of methods for detection of peanut allergens * SYBR Green ** 1 2 3 4 5 6 7 8 Hershey's Peanut Butter Toast & Peanut Butter Trail Mix Tropical Blend Skippy: Creamy Peanut Butter * ** DNA 3 10 3 copies DNA <3 10 3 3
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