Foods

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12 pages, 3056 KiB

Open AccessArticle

An Accurate, Rapid and Cost-Effective Method for T-nos Detection Based on CRISPR/Cas12a

by Yuling Wang, Cheng Peng, Lin Ding, Zhixun Su, Xiaoyun Chen, Xiaofu Wang, Meihao Sun and Junfeng Xu

Foods 2023, 12(3), 615; https://doi.org/10.3390/foods12030615 - 01 Feb 2023

Cited by 6 | Viewed by 1817

Abstract

CRISPR/Cas12a technology is used for nucleic acid detection due to its specific recognition function and non-specific single-stranded DNA cleavage activity. Here, we developed a fluorescence visualisation detection method based on PCR and CRISPR/Cas12a approaches. The method was used to detect the nopaline synthase [...] Read more.

CRISPR/Cas12a technology is used for nucleic acid detection due to its specific recognition function and non-specific single-stranded DNA cleavage activity. Here, we developed a fluorescence visualisation detection method based on PCR and CRISPR/Cas12a approaches. The method was used to detect the nopaline synthase terminator (T-nos) of genetically modified (GM) crops, circumventing the need for expensive instruments and technicians. For enhanced sensitivity and stability of PCR-CRISPR/Cas12a detection, we separately optimised the reaction systems for PCR amplification and CRISPR/Cas12a detection. Eleven samples of soybean samples were assessed to determine the applicability of the PCR-CRISPR/Cas12a method. The method could specifically detect target gene levels as low as 60 copies in the reaction within 50 min. In addition, accurate detection of all 11 samples confirmed the applicability. The method is not limited by large-scale instruments, making it suitable for mass detection of transgenic components in plants in the field. In conclusion, we developed a new, accurate, rapid, and cost-effective method for GM detection. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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9 pages, 867 KiB

Open AccessCommunication

Detection and Quantification of Adulterated Beef and Mutton Products by Multiplex Droplet Digital PCR

by Chuan He, Lan Bai, Yifan Chen, Wei Jiang, Junwei Jia, Aihu Pan, Beibei Lv and Xiao Wu

Foods 2022, 11(19), 3034; https://doi.org/10.3390/foods11193034 - 30 Sep 2022

Cited by 6 | Viewed by 1508

Abstract

In order to seek high profit, businesses mix beef and mutton with cheap meat, such as duck, pork, and chicken. Five pairs of primers were designed for quintuple droplet digital PCR (qddPCR) of specific genomic regions from five selected species and specificity and [...] Read more.

In order to seek high profit, businesses mix beef and mutton with cheap meat, such as duck, pork, and chicken. Five pairs of primers were designed for quintuple droplet digital PCR (qddPCR) of specific genomic regions from five selected species and specificity and amplification efficiency were determined. The mixed DNA template with an equal copy number was used for detecting the accuracy and limit of multiplex PCR. The results showed that the primers and probes of the five selected species had good specificity with the minimum number of detection copies: 0.15 copies/µL beef (Bos taurus), 0.28 copies/μL duck (Anas platyrhynchos), 0.37 copies/μL pork (Sus scrofa), 0.39 copies/μL chicken (Gallus gallus), and 0.41 copies/μL mutton (Ovis aries), respectively. The five sets of primers and probes could quickly judge whether the specified meat components existed in the food commodities. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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13 pages, 1479 KiB

Open AccessArticle

Establishment and Validation of Reference Genes of Brassica napus L. for Digital PCR Detection of Genetically Modified Canola

by Likun Long, Zhenjuan Xing, Yuxuan He, Wei Yan, Congcong Li, Wei Xia, Liming Dong, Ning Zhao, Yue Ma, Yanbo Xie, Na Liu and Feiwu Li

Foods 2022, 11(16), 2535; https://doi.org/10.3390/foods11162535 - 22 Aug 2022

Cited by 2 | Viewed by 2258

Abstract

As an effective tool for genetically modified organism (GMO) quantification in complex matrices, digital PCR (dPCR) has been widely used for the quantification of genetically modified (GM) canola events; however, little is known about the quantification of GM canola events using endogenous reference [...] Read more.

As an effective tool for genetically modified organism (GMO) quantification in complex matrices, digital PCR (dPCR) has been widely used for the quantification of genetically modified (GM) canola events; however, little is known about the quantification of GM canola events using endogenous reference gene (ERG) characteristics by dPCR. To calculate and quantify the content of GM canola using endogenous reference gene (ERG) characteristics, the suitability of several ERGs of canola, such as cruciferin A (CruA), acetyl-CoA carboxylase (BnAcc), phosphoenolpyruvate carboxylase (PEP), cruciferin storage (BnC1), oleoyl hydrolase (Fat(A)), and high-mobility-group protein I/Y (HMG-I/Y), was investigated by droplet dPCR. BnAcc and BnC1 were more specific and stable in copy number in the genome of Brassica napus L. than the other genes. By performing intra-laboratory validation of the suitability of ERG characteristics for the quantification of GM canola events, the ddPCR methods for BnAcc and BnC1 were comprehensively demonstrated in dPCR assays. The methods could provide technical support for GM labeling regulations. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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11 pages, 1307 KiB

Open AccessArticle

Rapid On-Site Identification for Three Arcidae Species (Anadara kagoshimensis, Tegillarca granosa, and Anadara broughtonii) Using Ultrafast PCR Combined with Direct DNA Extraction

by Ga-Young Lee, Eiseul Kim, Seung-Min Yang and Hae-Yeong Kim

Foods 2022, 11(16), 2449; https://doi.org/10.3390/foods11162449 - 14 Aug 2022

Cited by 3 | Viewed by 1501

Abstract

Granular ark (Tegillarca granosa), broughton’s ribbed ark (Anadara broughtonii), and half-crenate ark (Anadara kagoshimensis) are important fishery resources throughout Asia; granular ark exhibiting a higher economic value due to its rarity. However, due to the similar morphological [...] Read more.

Granular ark (Tegillarca granosa), broughton’s ribbed ark (Anadara broughtonii), and half-crenate ark (Anadara kagoshimensis) are important fishery resources throughout Asia; granular ark exhibiting a higher economic value due to its rarity. However, due to the similar morphological characteristics of the three species, the less valuable species could be exploited for food fraud. In this study, we developed a rapid on-site identification method based on a microfluidic chip for the detection of the three ark shell species. We designed new species-specific primers, targeting the genes encoding mitochondrial cytochrome b or cytochrome c oxidase I, for the identification of the three ark shells and estimated their specificity against 17 species, which amplified only the target species. The sensitivity of each primer was 0.001 ng. In addition, this method was further improved to develop a direct ultrafast polymerase chain reaction (PCR) for on-site food monitoring, which would allow for completing the entire procedure (from sampling to obtaining the results) within 25 min without DNA extraction. Our direct, ultrafast PCR was successfully applied to differentiate the three species from 29 commercial products. Therefore, this assay could be used as a rapid and cost-effective approach for the on-site identification of ark shells in commercial food products. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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16 pages, 2971 KiB

Open AccessArticle

Visual Detection of Chicken Adulteration Based on a Lateral Flow Strip-PCR Strategy

by Haoyi Xu, Hangzhen Lan, Daodong Pan, Junfeng Xu and Xiaofu Wang

Foods 2022, 11(15), 2351; https://doi.org/10.3390/foods11152351 - 05 Aug 2022

Cited by 7 | Viewed by 2356

Abstract

The aim of this study was to develop an accurate, easy-to-use, and cost-effective method for the detection of chicken adulteration based on polymerase chain reaction (PCR) and lateral flow strip (LFS). We compared six DNA extraction methods, namely the cetyltrimethylammonium bromide (CTAB) method, [...] Read more.

The aim of this study was to develop an accurate, easy-to-use, and cost-effective method for the detection of chicken adulteration based on polymerase chain reaction (PCR) and lateral flow strip (LFS). We compared six DNA extraction methods, namely the cetyltrimethylammonium bromide (CTAB) method, salt method, urea method, SDS method, guanidine isothiocyanate method, and commercial kit method. The chicken cytb gene was used as a target to design specific primers. The specificity and sensitivity of the PCR-LFS system were tested using a self-assembled lateral flow measurement sensor. The results showed that the DNA concentration obtained by salt methods is up to 533 ± 84 ng µL⁻¹, is a suitable replacement for commercial kits. The PCR-LFS method exhibits high specificity at an annealing temperature of 62 °C and does not cross-react with other animal sources. This strategy is also highly sensitive, being able to detect 0.1% of chicken in artificial adulterated meat. The results of the test strips can be observed with the naked eye within 5 min, and this result is consistent with the electrophoresis result, demonstrating its high accuracy. Moreover, the detection system has already been successfully used to detect chicken in commercial samples. Hence, this PCR-LFS strategy provides a potential tool to verify the authenticity of chicken. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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12 pages, 2239 KiB

Open AccessArticle

Quantitative PCR Assays for the Strain-Specific Identification and Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus X253

by Lei Zhao, Dong Zhang, Yang Liu, Yi-Nan Zhang, Dong-Qing Meng, Qiong Xu, Jiang Zhong, Qiu-Yue Jiang, Yu Zhao and Shi-Jie Wang

Foods 2022, 11(15), 2282; https://doi.org/10.3390/foods11152282 - 30 Jul 2022

Cited by 5 | Viewed by 1819

Abstract

Probiotics are universally recognized for their health benefits, despite the fact that their effects depend on the strain. Identification and enumeration of probiotic strains are required prior to evaluating their effectiveness. Lacticaseibacillus rhamnosus X253 is a potential probiotic strain with antioxidant capacity. Comparative [...] Read more.

Probiotics are universally recognized for their health benefits, despite the fact that their effects depend on the strain. Identification and enumeration of probiotic strains are required prior to evaluating their effectiveness. Lacticaseibacillus rhamnosus X253 is a potential probiotic strain with antioxidant capacity. Comparative genomics and single nucleotide polymorphisms (SNPs) were used to identify a strain-specific locus within the holA gene for strain X253 that was distinct in 30 different L. rhamnosus strains. Using quantitative PCR, the primers and probe designed for the locus were able to distinguish L. rhamnosus X253 from the other 20 probiotic strains. The chosen locus remained stable over 19 generations. The sensitivity of the assay was 0.2 pg genomic DNA of L. rhamnosus X253, or 10³ cfu/mL bacteria of this strain. In terms of repeatability and reproducibility, relative standard deviations (RSD) were less than 1% and 3%, respectively. Additionally, this assay achieved accurate enumerations of L. rhamnosus X253 in spiked milk and complex powder samples. The strain-specific assay could be used for quality control and compliance assessment of dairy products. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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12 pages, 6483 KiB

Open AccessArticle

Development and Application of a Visual Duck Meat Detection Strategy for Molecular Diagnosis of Duck-Derived Components

by Xiaoyun Chen, Huiru Yu, Yi Ji, Wei Wei, Cheng Peng, Xiaofu Wang, Xiaoli Xu, Meihao Sun and Junfeng Xu

Foods 2022, 11(13), 1895; https://doi.org/10.3390/foods11131895 - 26 Jun 2022

Cited by 4 | Viewed by 1718

Abstract

To make meat adulteration detection systems faster, simpler and more efficient, we established a duck-derived meat rapid detection Recombinase Polymerase Amplification (dRPA) method by using interleukin 2 (IL-2) from nuclear genomic DNA as the target gene to design specific primers. We tested the [...] Read more.

To make meat adulteration detection systems faster, simpler and more efficient, we established a duck-derived meat rapid detection Recombinase Polymerase Amplification (dRPA) method by using interleukin 2 (IL-2) from nuclear genomic DNA as the target gene to design specific primers. We tested the dRPA detection system by comparing its sensitivity and specificity using real-time fluorescent PCR technology. By adjusting the ratio of reagents, this method shortens the time of DNA extraction and visualizes results in combination with colloidal gold immunoassay strips. Our results demonstrate that this dRPA method could specifically detect duck-derived components with a sensitivity of up to 23 copies/μL and the accuracy of the results is consistent with real-time fluorescent PCR. Additionally, dRPA can detect at least 1% of the duck meat content by mixing beef and mutton with duck meat in different proportions, which was verified by spot-check market samples. These results can be visualized with colloidal gold immunoassay strips with the same accuracy as real-time fluorescent RPA. dRPA can complete detection within 30 min, which shortens existing detection time and quickly visualizes the detection results on-site. This lays the groundwork for future large-scale standardized duck origin detection. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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11 pages, 1912 KiB

Open AccessArticle

Digital PCR-Based Characterization of a g10evo-epsps Gene-Specific Matrix Reference Material for Its Food and Feed Detection

by Xiaoyun Chen, Huiru Yu, Pengfei Wang, Cheng Peng, Xiaofu Wang, Xiaoli Xu, Junfeng Xu, Jingang Liang and Liang Li

Foods 2022, 11(13), 1888; https://doi.org/10.3390/foods11131888 - 25 Jun 2022

Cited by 1 | Viewed by 1679

Abstract

g10evo-epsps is a novel glyphosate herbicide-resistant gene that has been transferred to various crops such as soybean, corn, cotton, and rice. Here, we developed a gene-specific digital Polymerase Chain Reaction (dPCR) detection method for absolute quantitative analysis of g10evo-epsps, and characterized g10evo-epsps [...] Read more.

g10evo-epsps is a novel glyphosate herbicide-resistant gene that has been transferred to various crops such as soybean, corn, cotton, and rice. Here, we developed a gene-specific digital Polymerase Chain Reaction (dPCR) detection method for absolute quantitative analysis of g10evo-epsps, and characterized g10evo-epsps certified reference materials (CRM) using ZUTS-33 soybean powder as the candidate material. Stability tests of matrix CRMs demonstrate that these CRMs can be stored stably for 6 months and transported for 10 days at room temperature and withstand summer high temperatures (below 60 °C). CRM characterization is based on the copy number ratio of g10evo-epsps to lectin. Eight qualified laboratories independently validated the CRM using dPCR method, with a measurement of 0.98 (copy/copy) and an extended uncertainty of 0.08 (copy/copy). The g10evo-epsps matrix CRM described here may be used for qualitative and quantitative testing, method evaluation, laboratory quality control, and other related fields. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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13 pages, 1356 KiB

Open AccessArticle

Collaborative Ring Trial of the Applicability of a Reference Plasmid DNA Calibrant in the Quantitative Analysis of GM Maize Event MON810

by Yanan Meng, Shu Wang, Jinchao Guo and Litao Yang

Foods 2022, 11(11), 1538; https://doi.org/10.3390/foods11111538 - 24 May 2022

Viewed by 1283

Abstract

Certified reference materials (CRMs) is one of the critical requirements in a quantitative analytical method, such as in the quantification of genetically modified (GM) contents in food/feed products. Plasmid-DNA-based CRMs are becoming essential in GM content quantification. Herein, we report the construction of [...] Read more.

Certified reference materials (CRMs) is one of the critical requirements in a quantitative analytical method, such as in the quantification of genetically modified (GM) contents in food/feed products. Plasmid-DNA-based CRMs are becoming essential in GM content quantification. Herein, we report the construction of one plasmid DNA calibrant, pMON810, for the quantification of the GM maize event MON810 which is commercially planted and used for food/feeds worldwide, and the collaborative ring trial was used to validate its applicability. pMON10 was proven to have high specificity for the MON810 event. The limit of detection (LOD) and limit of quantification (LOQ) of real-time PCR assays of MON810 event and maize endogenous gene using pMON810 as calibrant was 2 copies/μL and 5 copies/μL, respectively. A total of eight laboratories participated in the ring trial and returned valid test results. Each sample was performed with three repeats and three parallels in each repeat. Statistical analysis of the ring trial results showed that pMON810 as a calibrant had high PCR efficiency (ranging from 0.885 to 1.008) and good linearity (ranging from 0.9933 to 0.9997) in MON810 and endogenous gene real-time PCR assays. The bias between the test values and true values ranged from 4.60 to 20.00% in the quantification of five blind samples. These results indicate that pMON810 is suitable for use as a calibrant for the quantification of MON810 events in routine lab analysis or to evaluate detection methods for MON810, as well as being used as a substitute for the matrix-based CRM of MON810. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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11 pages, 1267 KiB

Open AccessArticle

Comparison of Real-Time PCR and Droplet Digital PCR for the Quantitative Detection of Lactiplantibacillus plantarum subsp. plantarum

by Chang-Hun Choi, Eiseul Kim, Seung-Min Yang, Da-Som Kim, Seung-Man Suh, Ga-Young Lee and Hae-Yeong Kim

Foods 2022, 11(9), 1331; https://doi.org/10.3390/foods11091331 - 03 May 2022

Cited by 8 | Viewed by 3158

Abstract

Droplet digital polymerase chain reaction (ddPCR) is one of the newest and most promising tools providing absolute quantification of target DNA molecules. Despite its emerging applications in microorganisms, few studies reported its use for detecting lactic acid bacteria. This study evaluated the applicability [...] Read more.

Droplet digital polymerase chain reaction (ddPCR) is one of the newest and most promising tools providing absolute quantification of target DNA molecules. Despite its emerging applications in microorganisms, few studies reported its use for detecting lactic acid bacteria. This study evaluated the applicability of a ddPCR assay targeting molecular genes obtained from in silico analysis for detecting Lactiplantibacillus plantarum subsp. plantarum, a bacterium mainly used as a starter or responsible for fermentation in food. The performance characteristics of a ddPCR were compared to those of a quantitative real-time PCR (qPCR). To compare the linearity and sensitivity of a qPCR and ddPCR, the calibration curve for a qPCR and the regression curve for a ddPCR were obtained using genomic DNA [10²–10⁸ colony-forming units (CFU)/mL] extracted from a pure culture and spiked food sample. Both the qPCR and ddPCR assays exhibited good linearity with a high coefficient of determination in the pure culture and spiked food sample (R² ≥ 0.996). The ddPCR showed a 10-fold lower limit of detection, suggesting that a ddPCR is more sensitive than a qPCR. However, a ddPCR has limitations in the absolute quantitation of high bacterial concentrations (>10⁶ CFU/mL). In conclusion, a ddPCR can be a reliable method for detecting and quantifying lactic acid bacteria in food. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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11 pages, 1518 KiB

Open AccessArticle

Development of an Event-Specific Droplet Digital PCR Assay for Quantification and Evaluation of the Transgene DNAs in Trace Samples of GM PRNP-Knockout Goat

by Wenting Xu, Ping Shen, Rong Li, Biao Liu and Litao Yang

Foods 2022, 11(6), 868; https://doi.org/10.3390/foods11060868 - 18 Mar 2022

Cited by 2 | Viewed by 2113

Abstract

The prion protein (PRNP) gene encoding prion protein is considered a prerequisite for the occurrence of scrapie disease, and knockout of the PRNP gene in transgenic goat is one effective approach to avoid scrapie. This study aims to establish an event-specific [...] Read more.

The prion protein (PRNP) gene encoding prion protein is considered a prerequisite for the occurrence of scrapie disease, and knockout of the PRNP gene in transgenic goat is one effective approach to avoid scrapie. This study aims to establish an event-specific droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify the content of genetically modified (GM) PRNP-knockout goat event KoP1. The developed ddPCR assay presents high specificity, sensitivity, accuracy, precision and wide dynamic range. The limits of detection and quantification were as low as 1.44 and 7.2 haploid genome equivalent (HGE) per reaction, respectively. Furthermore, this assay was successfully applied in quantifying the goat KoP1 GM content in milk, feces and living environmental soil samples. We believe that the developed ddPCR assay has the potential to be used in the evaluation of horizontal gene transfer and the practical risk assessment of GM goat event KoP1 and its derivatives. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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Review

Jump to: Research, Other

19 pages, 2120 KiB

Open AccessReview

Review of CRISPR/Cas Systems on Detection of Nucleotide Sequences

by Mengyu Wang, Haoqian Wang, Kai Li, Xiaoman Li, Xujing Wang and Zhixing Wang

Foods 2023, 12(3), 477; https://doi.org/10.3390/foods12030477 - 19 Jan 2023

Cited by 4 | Viewed by 2881

Abstract

Nowadays, with the rapid development of biotechnology, the CRISPR/Cas technology in particular has produced many new traits and products. Therefore, rapid and high-resolution detection methods for biotechnology products are urgently needed, which is extremely important for safety regulation. Recently, in addition to being [...] Read more.

Nowadays, with the rapid development of biotechnology, the CRISPR/Cas technology in particular has produced many new traits and products. Therefore, rapid and high-resolution detection methods for biotechnology products are urgently needed, which is extremely important for safety regulation. Recently, in addition to being gene editing tools, CRISPR/Cas systems have also been used in detection of various targets. CRISPR/Cas systems can be successfully used to detect nucleic acids, proteins, metal ions and others in combination with a variety of technologies, with great application prospects in the future. However, there are still some challenges need to be addressed. In this review, we will list some detection methods of genetically modified (GM) crops, gene-edited crops and single-nucleotide polymorphisms (SNPs) based on CRISPR/Cas systems, hoping to bring some inspiration or ideas to readers. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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Other

Jump to: Research, Review

9 pages, 1212 KiB

Open AccessPerspective

PCR Mediated Nucleic Acid Molecular Recognition Technology for Detection of Viable and Dead Foodborne Pathogens

by Mengtao Chen, Xinyue Lan, Longjiao Zhu, Ping Ru, Wentao Xu and Haiyan Liu

Foods 2022, 11(17), 2675; https://doi.org/10.3390/foods11172675 - 02 Sep 2022

Cited by 10 | Viewed by 2269

Abstract

Living foodborne pathogens pose a serious threat to public and population health. To ensure food safety, it is necessary to complete the detection of viable bacteria in a short time (several hours to 1 day). However, the traditional methods by bacterial culture, as [...] Read more.

Living foodborne pathogens pose a serious threat to public and population health. To ensure food safety, it is necessary to complete the detection of viable bacteria in a short time (several hours to 1 day). However, the traditional methods by bacterial culture, as the gold standard, are cumbersome and time-consuming. To break through the resultant research bottleneck, PCR mediated nucleic acid molecular recognition technologies, including RNA-based reverse transcriptase PCR (RT-PCR) and DNA-based viability PCR (vPCR) have been developed in recent years. They not only sensitively amplify detection signals and quickly report detection results, but also distinguish viable and dead bacteria. Therefore, this review introduces these PCR-mediated techniques independent of culture for viable and dead foodborne pathogen detection from the nucleic acid molecular recognition principal level and describes their whole-process applications in food quality supervision, which provides a useful reference for the development of detection of foodborne pathogens in the future. Full article

(This article belongs to the Special Issue PCR in Food Science: Current Technology and Applications)

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