| The incidence rate of incidence of food allergy has been increasing in recent years.It is reported that the incidence rate of global food allergy is 4-6%,and the incidence rate of food allergy among children under 9 years of age in Europe and the United States is 7%-8%.More than 95%of children's food allergies are related to milk,eggs,peanuts,soybeans,wheat and fish,while adults are usually related to peanuts,nuts,fish and shells.In order to detect allergens in food quickly,a real-time fluorescent quantitative PCR method with triple TaqMan probe was established.1.A real-time fluorescent quantitative PCR method was established for the detection of allergen genes in peanut,soybean and walnut.Using real-time quantitative PCR(qPCR)technology,TaqMan probe rapid detection method was established for common allergens,such as soybean allergen protein gene(Gly m Bd28k),walnut allergen gene(Jug r 1),peanut allergen gene(Ara h 1).After searching and comparing the allergen genes of three species of soybean,walnut and peanut in GenBank,a pair of specific primers and probes with different fluorescence groups were designed respectively,and a fluorescence quantitative detection method of single TaqMan probe for allergen genes of soybean,walnut and peanut was established.The conditions of annealing temperature,primer,probe concentration and reaction system were optimized respectively,and the reactants were obtained The specificity,sensitivity and repeatability tests were carried out to verify the results.The results showed that the specificity of single real-time fluorescent quantitative PCR for soybean,walnut and peanut was good,and only the corresponding target fragment could be amplified specifically.The standard curve was established.The correlation coefficient:soybean:R~2=0.996,walnut:R~2=0.995,peanut:R~2=0.999,amplification efficiency:soybean:E=106.4%,walnut:E=109.5%,peanut:E=105.4%;the minimum detection limit of plasmid,soybean:1.50×10~0 copies/?L,walnut:1.50×10~1copies/?L?peanut:1.50×10~2copies/?L The detection limit of DNA mixed sample:soybean,walnut and peanut:0.1ng/?L;the detection threshold of allergen increased with the increase of temperature or time;the coefficient of variation was less than 2%.2.Double fluorescence quantitative PCR was established to detect the allergen genes of soybean and walnut.The results of standard curve showed that correlation coefficient:soybean:R~2=0.993,walnut:R~2=0.999,amplification efficiency:soybean:E=104.9%,walnut:E=98.0%.The results of specificity test showed that only the DNA of soybean and walnut could be detected.The lowest detection limit of this method was soybean:1.50×10~3 copies/?L,walnut:1.50×10~2 copies/?L,the minimum detection limit of DNA mixed samples is 0.01ng/?L;the repeatability test results show that the coefficient of variation of repeatability within and between groups is within 2%.The detection results of rough processed samples show that the detection threshold of allergens increases with the increase of temperature or time.3.A real-time fluorescence quantitative PCR method was established to detect soybean,walnut and peanut.The results of standard curve showed that the correlation coefficient:soybean:R~2=0.993,walnut:R~2=0.991,peanut:R~2=0.997,amplification efficiency:soybean:E=106.9%,walnut:E=112.1%,peanut:E=108.9%;the specificity of this method was good,only the related genes could be amplified specifically,and the lowest copy number of the real-time fluorescent quantitative PCR method was1.50×10~2,walnut:1.50×10~2 copies/?L,peanut:1.50×10~3copies/?L,The lowest detection limits of DNA mixed samples were soybean:0.01ng/?L,walnut:0.1ng/?L,peanut:1ng/?L.The repeatability test showed that the coefficient of variation of the repeatability between and within groups was within 2%,which indicated that the established triple fluorescence quantitative PCR method had good repeatability;the detection results of rough processed samples showed that the detection threshold of allergens increased with the increase of temperature or time.The method was used to detect 50 processed foods,among which,there were positive amplification signals in the samples containing soybean,walnut and peanut allergens.This study successfully established a real-time fluorescence quantitative PCR detection method for the triple TaqMan probe of allergen genes in soybean,walnut and peanut.It has the characteristics of high specificity and low detection limit through rapid detection and verification in the market processed food. |
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