Food Research And Development. Ara h1 GenBank PCR PCR -CT PCR PCR

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1 Food Research And Development Ara h1 * Ara h1 GenBank Ara h1 DNA AF SYBR Green -CT 2 8 Ara h1 SYBR Green copies R copies Ara h1 Ara h %<5 % ~0.4 % 3 3 Table 3 Precision test A Development of Two Assays to Detect Peanut Allergen Ara h1 DNA Residue in Food Products Table RSD/% % 2.6 % 5 ml50 ml 0.1 ml g/l 4 CHEN Jia-jieWANG Hai-yanLiang Qiu-ni JI Kun-meiLIU Zhi-gang * * 4 Determination results of recovery /mg/l /mg/l /mg/l /% /% RSD/% % 1 20g/mL 15 min [1]. [J].,2006(1): [2]. [J].,1993,24 (10): [3]. [J ].,2004(5): [4]. [J]. 2007(3)

2 70 Ara h1 State Key Laboratory of Respiratory Disease for Allergy at Shengzhen UniversityShenzhen UniversityShenzhen GuangdongChina 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.AF 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 to copies with of R 2 value and 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 h [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] GenBank No.AF DNA DNA CTAB [8] [7] Real-time 8 DNA DNA OD 260 /OD DNA Ara h 1 DNA Ara h1 Ara h1 OF1Ara h1 OR1 DNA DNA Ara h1 IF2Ara h1 IR2 DNA 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 min

3 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 s s 72 1 min min pmd-ara h1at/a pmd18-t pmd-ara h1a E.coli JM copies/μl 109 LB Ct BamHIHindIII Ara h1 DNA DNA DNA CTAB [8] 8 DNA 50 ng/μl 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 DNA Prism 7300 Applied Biosystems, Warrington, UK min, s, 57 1 min 1 CTAB 8 [9] Vaǐtilingom M DNA DNA 85 ng/μl ~250 ng/μl, OD 260 / OD ~ 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 ~1.75 DNA Ara h1 DNA SYBR Green Ara h1 DNA ABI Prism Ara h1 DNA 1 2

4 72 Ara h1 3 DNA 4 GenBank pmd-ara h1a bp bp bp bp bp 500 bp M DNA Maker DL DNA Fig.1 Analysis of the genomic DNA of various foods bp bp 750 bp 500 bp 250 bp 100 bp 2 1 DNA bp bp 2 DNA M DNA Maker BamH Hind Fig.3 Restriction analysis for standard template vector plasmids with BamHand Hind 8 Ara h1 DNA bp 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 DL DNA Fig.2 Analysis of -products of the genomic DNA from peanut by nested 2.4 SYBR Green DNA CT CT 2.3 pmd-ara h1a 1 pmd18-t 5 CT DNA BamH/Hind CT 350 bp CT

5 Ara h pmd-ara h1a DNA Fig.5 Amplification curve of real time with series of dilutions of standard template pmd-ara h1a copies CT copies R 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 copies DNA CTAB DNA Ara h1 DNA Two kinds of methods for detection of peanut allergens * SYBR Green ** Hershey's Peanut Butter Toast & Peanut Butter Trail Mix Tropical Blend Skippy: Creamy Peanut Butter * ** DNA copies DNA <

6 74 Ara h FDA [10] Allergy Immunol, 1999, 10(l):14-20 FDA 1/4 [11] (4): [4]. Ara h1 Ara h2 665 DNA Arah1 190 comparison[j]. Eur J Public Health 2008, 18(2): DNA [12] Ara h1dna science and food safety, 2007, 6(2):47-58 Elena Scaravelli Ara h3 Food control 2002,13(4): [13] R-Biopharm [7] Ara h2 SYBR Green [1] Warner JO. Peanut allergy: a major public health issue [J]. Pediatr [2] Allan S, Anne MFHugh AS. Fatalities due to anaphylactic reactions to foods[j]. J Allergy Clin Immunol, 2001, 107(1): [3] Allan S, Anne MFHugh AS. Further fatalities caused by anaphylactic reactions to food, [J]. J Allergy Clin Immunol2007,119 DNA 47(12): [11] (6) Ara h1 DNA DNA (3): copies 8 food products[j]. Eur Food Res Technol 2 [J] (8): [5] Steve LTSue LH. Food allergen labeling in the USA and Europe[J]. Current opinion in allergy and clinical immunology2006,6(3):186- [6] Cornelisse -Vermaat JR, Voordouw J, Yiakoumaki V, et al. Food - allergic consumers labeling preferences: a cross -cultural [7] Wen HW, Wlodzimierz BW, Thomas RDet al. Peanut Allergy, Peanut Allergens, and Methods for the Detection of Peanut Contamination in Food Products[J]. Comprehensive reviews in food [8] Alary R, Serin A, Maury D. Comparison of simplex and duplex realtime for the quantification of GMO in maize and soybean [J]. [9] Vaitilingom M, Pijinenburg H, Gendre F, et al. Real -time detection of genetically modified maximizer maize and round up ready soybean in some representative foods [J]. Food chemistry 1999, [10] [J]. 30(5) [J] [12] Hird H, Lloyd J, Goodier Ret al. Detection of peanut using realtime polymerase chain reaction[j]. Eur Food Res Technol2003,217 [13] Scaravelli E, Brohee M, Marchelli Ret al. Development of three real-time assays to detect peanut allergen residue in processed. 2008, 227(3):

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