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Rapid on-site detection of shrimp allergen tropomyosin using a novel ultrafast PCR system.

ABSTRACT: Shrimp is seafood that can commonly trigger allergic reactions. In this study, the ultrafast real-time PCR assay with portable device was developed to detect a shrimp-derived major allergen, tropomyosin, without complicated DNA extraction. For shrimp allergen detection, a specific primer pair was designed based on the shrimp tropomyosin gene and 18S ribosomal RNA gene as internal control. Primer specificity was assessed using 8 common seafood species. Serially diluted shrimp DNA was used to determine the limit of detection of the ultrafast PCR system, which was approximately 3.2 pg. Twenty-three food samples containing shrimp were evaluated to verify the applicability of a direct ultrafast PCR method for detecting shrimp allergens without DNA isolation. It took less than 30 min from sample preparation-to-result analysis to detect shrimp DNA in raw and processed samples. Therefore, this PCR system can be effectively and conveniently utilized in the field to detect shrimp in various food products.

SUBMITTER: Kim MJ

PROVIDER: S-EPMC6431315 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Rapid on-site detection of shrimp allergen tropomyosin using a novel ultrafast PCR system.

Kim Mi-Ju MJ Kim Hee-In HI Kim Jae-Hwan JH Suh Seung-Man SM Kim Hae-Yeong HY

Food science and biotechnology 20180927 2

Shrimp is seafood that can commonly trigger allergic reactions. In this study, the ultrafast real-time PCR assay with portable device was developed to detect a shrimp-derived major allergen, tropomyosin, without complicated DNA extraction. For shrimp allergen detection, a specific primer pair was designed based on the shrimp tropomyosin gene and 18S ribosomal RNA gene as internal control. Primer specificity was assessed using 8 common seafood species. Serially diluted shrimp DNA was used to dete ...[more]

PMID: 30956872

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Project description:BackgroundDetection of Plasmodium spp. is sometimes inconvenient especially in rural areas that are distant from a laboratory. In this study a portable diagnostic test of Plasmodium spp. was developed using insulated isothermal polymerase chain reaction (iiPCR) as an alternative approach to improve this situation.MethodsA pair of universal primers and probe were designed to amplify and detect gene encoding 18S small sub-unit rRNA of Plasmodium spp using iiPCR method in a portable device, POCKIT™. The efficiency and detection limit of the assay were evaluated using quantitative real-time polymerase chain reaction (qPCR) approach before being subjected to testing in POCKIT™. Detection results of POCKIT™ were displayed as '+', '-' or '?' based on the fluorescence ratio after/before reaction. A total of 55 and 35 samples from malaria patients and healthy subjects, respectively, were screened to evaluate the feasibility of this newly designed iiPCR assay.ResultsThe iiPCR assay allowed the detection of various species of Plasmodium, including those infecting humans (Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae, P. ovale), monkeys, birds, and rodents. Efficiency of the assay achieved 96.9 % while the lower detection limit was ≥100 copies of plasmodial DNA. Specificity of the assay was assured as it could not detect human, bacterial and other parasitic DNA. Among the 55 clinical samples tested, 47 (85.4 %) of them were detected as positive by POCKIT™. Four (7.3 %) samples with fluorescence ratio after/before reaction of <1.2 were reported as negative while another four (7.3 %) were ambiguously detected as they had fluorescence ratios between 1.2 and 1.3. The fluorescence ratio was not found to be associated with the copy number of plasmodial DNA. This approach can only be considered as a qualitative method.ConclusionsThe portable iiPCR system may serve as an alternative approach for preliminary screening of malaria in endemic rural areas. The system may also be useful for detecting animal malaria in the field. Although it is not as quantitative as qPCR method, it is comparatively fast and easy to handle. It is believed that the POCKIT-iiPCR assay is able to achieve 100 % sensitivity if increased amount of DNA from each sample is used. The iiPCR assay can also be upgraded in future to detect multiple Plasmodium spp. at the same time by designing the specific primers and probes.

Dataset Information

Rapid on-site detection of shrimp allergen tropomyosin using a novel ultrafast PCR system.

Publications

Rapid on-site detection of shrimp allergen tropomyosin using a novel ultrafast PCR system.

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