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Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify...

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Main Authors: Asing, Md. Eaqub Ali, Abd Hamid, Sharifah Bee, Hossain, M.A. Motalib, Mustafa, Shuhaimi, Abdul Kader, Md., Sarker, Md. Zaidul Islam
Format: Article
Language:English
English
Published: PLOS One Publishing 2016
Subjects:
R Medicine (General)
Online Access:http://irep.iium.edu.my/53846/1/journal.pone.0163436.PDF
http://irep.iium.edu.my/53846/6/53846_Lab-on-a-chip-based%20PCR-RFLP_Scopus.pdf
http://irep.iium.edu.my/53846/
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163436
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spelling my.iium.irep.538462017-03-14T01:16:42Z http://irep.iium.edu.my/53846/ Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines Asing, Md. Eaqub Ali Abd Hamid, Sharifah Bee Hossain, M.A. Motalib Mustafa, Shuhaimi Abdul Kader, Md. Sarker, Md. Zaidul Islam R Medicine (General) The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition. PLOS One Publishing 2016-10-07 Article REM application/pdf en http://irep.iium.edu.my/53846/1/journal.pone.0163436.PDF application/pdf en http://irep.iium.edu.my/53846/6/53846_Lab-on-a-chip-based%20PCR-RFLP_Scopus.pdf Asing, Md. Eaqub Ali and Abd Hamid, Sharifah Bee and Hossain, M.A. Motalib and Mustafa, Shuhaimi and Abdul Kader, Md. and Sarker, Md. Zaidul Islam (2016) Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines. PLOS One, 11 (10). pp. 1-27. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163436 10.1371/journal.pone.0163436
institution Universiti Islam Antarabangsa Malaysia
building IIUM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider International Islamic University Malaysia
content_source IIUM Repository (IREP)
url_provider http://irep.iium.edu.my/
language English
English
topic R Medicine (General)
spellingShingle R Medicine (General)
Asing, Md. Eaqub Ali
Abd Hamid, Sharifah Bee
Hossain, M.A. Motalib
Mustafa, Shuhaimi
Abdul Kader, Md.
Sarker, Md. Zaidul Islam
Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines
description The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.
format Article
author Asing, Md. Eaqub Ali
Abd Hamid, Sharifah Bee
Hossain, M.A. Motalib
Mustafa, Shuhaimi
Abdul Kader, Md.
Sarker, Md. Zaidul Islam
author_facet Asing, Md. Eaqub Ali
Abd Hamid, Sharifah Bee
Hossain, M.A. Motalib
Mustafa, Shuhaimi
Abdul Kader, Md.
Sarker, Md. Zaidul Islam
author_sort Asing, Md. Eaqub Ali
title Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines
title_short Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines
title_full Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines
title_fullStr Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines
title_full_unstemmed Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines
title_sort lab-on-a-chip-based pcr-rflp assay for the detection of malayan box turtle (cuora amboinensis)in the food chain and traditional chinese medicines
publisher PLOS One Publishing
publishDate 2016
url http://irep.iium.edu.my/53846/1/journal.pone.0163436.PDF
http://irep.iium.edu.my/53846/6/53846_Lab-on-a-chip-based%20PCR-RFLP_Scopus.pdf
http://irep.iium.edu.my/53846/
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163436
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score 13.211869

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