Dataset Information

Simultaneous detection of fruit allergen-coding genes in tomato, apple, peach and kiwi through multiplex PCR.

ABSTRACT: Fruit allergies have become more common in recent years, and are now a serious health problem. In this study, a multiplex PCR assay was used to detect potential fruit allergens causing food allergy labeling in Korea. For the detection of these allergens, specific primer pairs were designed to amplify the allergen-coding genes Cyclophilin (tomato), Mdtl 1 (apple), Pru p 2.01A (peach) and Pectin methylesterase inhibitor (kiwi), and primer pair targeting the 18S ribosomal RNA gene was additionally used as an endogenous control. Primer specificity was assessed with 23 plant species. A mixture of DNA from the four fruits was serially diluted and used to determine the sensitivity of the multiplex PCR assay, which was approximately 0.08 ng. Eleven commercial fruit products were evaluated to verify the applicability of the multiplex PCR assay. This assay is expected to be a specific and efficient method for detecting fruit allergens in foods.

SUBMITTER: Suh SM

PROVIDER: S-EPMC6811467 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Simultaneous detection of fruit allergen-coding genes in tomato, apple, peach and kiwi through multiplex PCR.

Suh Seung-Man SM Park Saet-Byul SB Kim Mi-Ju MJ Kim Hae-Yeong HY

Food science and biotechnology 20190316 5

Fruit allergies have become more common in recent years, and are now a serious health problem. In this study, a multiplex PCR assay was used to detect potential fruit allergens causing food allergy labeling in Korea. For the detection of these allergens, specific primer pairs were designed to amplify the allergen-coding genes Cyclophilin (tomato), Mdtl 1 (apple), Pru p 2.01A (peach) and Pectin methylesterase inhibitor (kiwi), and primer pair targeting the 18S ribosoma ...[more]

PMID: 31695960

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Project description:BACKGROUND: Fruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis)Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica). RESULTS: Eight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01-3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2. CONCLUSION: A total of 18 putative peach/almond allergen genes have been mapped on five linkage groups. Their positions confirm the high macro-synteny between peach/almond and apple. The insight gained will help to identify key genes causing differences in allergenicity among different cultivars of peach and other Prunus species.

Project description:BackgroundPru p 1 is a major allergen in peach and nectarine, and the different content in varieties may affect the degree of allergic reactions. This study aimed to quantify Pru p 1 levels in representative peach varieties and select hypoallergenic Pru p 1 varieties.MethodsTo obtain monoclonal and polyclonal antibodies, mice and rabbits, respectively, were immunized with recombinant Pru p 1.01 and Pru p 1.02. The Pru p 1 levels in fruits from 83 representative peach varieties was quantified by sandwich enzyme-linked immunosorbent assay (sELISA). nPru p 1 was obtained through specific monoclonal antibody affinity purification and confirmed by Western blot and mass spectrometry. The variable Pru p 1 content of selected varieties was evaluated by Western blot and the expression level of encoding Pru p 1 genes by quantitative polymerase chain reaction.ResultsA sELISA method with monoclonal and polyclonal antibodies was built for quantifying Pru p 1 levels in peach. Pru p 1 was mainly concentrated in the peel (0.20-73.44 μg/g, fresh weight), being very low in the pulp (0.05-9.62 μg/g) and not detected in wild peach. For the 78 peach and nectarine varieties, Pru p 1 content varied widely from 0.12 to 6.45 μg/g in whole fruit. We verified that natural Pru p 1 is composed of 1.01 and 1.02 isoallergens, and the Pru p 1 expression level and Pru p 1 band intensity in the immunoblots were in agreement with protein quantity determined by ELISA for some tested varieties. In some cases, the reduced levels of Pru p 1 did not coincide with low Pru p 3 in the same variety in whole fruit, while some ancient wild peach and nectarines contained low levels of both allergens, and late-ripening yellow flesh varieties were usually highly allergenic.ConclusionPru p 1 content is generally low in peach compared to Pru p 3. Several hypoallergenic Pru p 1 and Pru p 3 varieties, "Zi Xue Tao," "Wu Yue Xian," and "May Fire," were identified, which could be useful in trials for peach allergy patients.

Project description:Malaysia is a tropical country that produces commercial fruits, including star fruits, Averrhoa carambola L. (Oxalidales: Oxalidaceae), and guavas, Psidium guajava L. (Myrtales: Myrtaceae). There is a high demand for these fruits, and they are planted for both local consumption and export purposes. Unfortunately, there has been a gradual reduction of these fruits, which has been shown to be related to fruit fly infestation, especially from the Bactrocera species. Most parasitic wasps (Hymenoptera: Braconidae: Opiinae) are known as parasitoids of fruit fly larvae. In this study, star fruits and guavas infested by fruit fry larvae were collected from the Malaysian Agricultural Research and Development Institute. The parasitized larvae were reared under laboratory conditions until the emergence of adult parasitoids. Multiplex PCR was performed to determine the braconid species using two mitochondrial DNA markers, namely cytochrome oxidase subunit I and cytochrome b. Two benefits of using multiplex PCR are the targeted bands can be amplified simultaneously using the same reaction and the identification process of the braconid species can be done accurately and rapidly. The species of fruit flies were confirmed using the COI marker. The results obtained from our study show that Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), Fopius arisanus (Sonan), and Pysttalia incisi (Silvestri) were parasitoids associated with Bactrocera carambolae (Drew and Hancock) (Diptera: Tephritidae) infested star fruits. Fopius arisanus was also the parasitoid associated with Bactrocera papayae (Drew and Hancock) infested guavas. Maximum parsimony was been constructed in Opiinae species to compare tree resolution between these two genes in differentiating among closely related species. The confirmation of the relationship between braconids and fruit fly species is very important, recognized as preliminary data, and highly necessary in biological control programs.

Dataset Information

Simultaneous detection of fruit allergen-coding genes in tomato, apple, peach and kiwi through multiplex PCR.

Publications

Simultaneous detection of fruit allergen-coding genes in tomato, apple, peach and kiwi through multiplex PCR.

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