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/ http://irep.iium.edu.my/53846/ http://irep.iium.edu.my/53846/ 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

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recordtype	eprints
spelling	iium-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 PeerReviewed 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
repository_type	Digital Repository
institution_category	Local University
institution	International Islamic University Malaysia
building	IIUM Repository
collection	Online Access
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/ http://irep.iium.edu.my/53846/ http://irep.iium.edu.my/53846/ 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
first_indexed	2023-09-18T21:16:10Z
last_indexed	2023-09-18T21:16:10Z
_version_	1777411576951734272

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

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