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Double gene targeting PCR assay for the detection of Crocodylus porosus in commercial products

Ahmad Nizar NN, Ali ME, Hossain MAM, Sultana S, Ahamad MNU

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2018 Jun;35(6):1038-1051.
PMID: 29447579 DOI: 10.1080/19440049.2018.1440644

The demand for crocodile meat is quickly growing because of its exotic and organoleptic appeal and also the low content of cholesterol and lipids. Moreover, crocodile oil and blood have been used in alternative medicines for treating asthma and several other ailments since ancient times. Furthermore, crocodile hides have great demand in leather industries. All of these have collectively contributed to the extensive hunting, illegal trading and consequent decline of crocodiles in most parts of the world. To keep space with the growing demands, some crocodile species such as Crocodylus porosus have been raised in farms and its commercial trades have been legalised. However, demand for wild crocodiles in foods and medicines has continued in high gear. Recently, several DNA-based methods have been proposed for crocodile detection, but those assays are based on single gene and longer-sized amplicon targets that break down during extensive processing. To address this gap, here we developed and validated a highly stable double gene targeted multiplex PCR assay for the identification of C. porosus materials in commercial products. The assay involved two short sites from C. porosus atp6 (77 bp) and cytb (127 bp) genes and a universal internal control (99 bp) for eukaryotes. The PCR primers were cross-tested against 18 species and validated under pure and mixed matrices under extensive boiling, autoclaving and microwave cooking conditions. Finally, it was used to identify five crocodile-based commercial products. The lower limits of detection for atp6 and cytb genes were 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% under mixed matrices. Some inherent features, such as 77-127 bp amplicon sizes, exceptional stability and superior sensitivity, suggested the assay could be used for the identification of C. porosus in any forensic specimen.

Matched MeSH terms: Mitochondrial Proton-Translocating ATPases/genetics*; Mitochondrial Proton-Translocating ATPases/metabolism
Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain

Ahamad MNU, Ali ME, Hossain MAM, Asing A, Sultana S, Jahurul MHA

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2017 Dec;34(12):2043-2057.
PMID: 28748739 DOI: 10.1080/19440049.2017.1359752

Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.

Matched MeSH terms: Mitochondrial Proton-Translocating ATPases/genetics; Mitochondrial Proton-Translocating ATPases/metabolism
Selection of reference genes for quantitative studies in acute myeloid leukaemia

Lee C, Yiau KXS, Lee LJ, Chong PP, Chang KM, Abdullah M

Malays J Pathol, 2019 Dec;41(3):313-326.
PMID: 31901916

INTRODUCTION: Quantitative polymerase chain reaction (qPCR) is commonly used in the investigation of acute myeloid leukaemias (AML). Stable reference genes (RG) are essential for accurate and reliable reporting but no standard method for selection has been endorsed.
MATERIALS AND METHODS: We evaluated simple statistics and published model-based approaches. Multiplex-qPCR was conducted to determine the expression of 24 candidate RG in AMLs (N=9). Singleplex-qPCR was carried out on selected RG (SRP14, B2M and ATP5B) and genes of interest in AML (N=15) and healthy controls, HC (N=12).
RESULTS: RG expression levels in AML samples were highly variable and coefficient of variance (CV) ranged from 0.37% to 10.17%. Analysis using GeNorm and Normfinder listed different orders of most stable genes but the top seven (ACTB, UBE2D2, B2M, NF45, RPL37A, GK, QARS) were the same. In singleplex-qPCR, SRP14 maintained the lowest CV in AML samples. B2M, one of most stable reference genes in AML, was expressed near significantly different in AML and HC. GeNorm selected ATP5B+SRP14 while Normfinder chose SRP14+B2M as the best two RG in combination. The median expressions of combined RG genes in AML compared to HC were less significantly different than individually implying smaller expression variation after combination. Genes of interest normalised with RG in combination or individually, displayed significantly different expression patterns.
CONCLUSIONS: The selection of best reference gene in qPCR must consider all sample sets. Model-based approaches are important in large candidate gene analysis. This study showed combination of RG SRP14+B2M was the most suitable normalisation factor for qPCR analysis of AML and healthy individuals.

Matched MeSH terms: Mitochondrial Proton-Translocating ATPases/genetics*

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