This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.
An efficient electrochemical impedance genosensing platform has been constructed based on graphene/zinc oxide nanocomposite produced via a facile and green approach. Highly pristine graphene was synthesised from graphite through liquid phase sonication and then mixed with zinc acetate hexahydrate for the synthesis of graphene/zinc oxide nanocomposite by solvothermal growth. The as-synthesised graphene/zinc oxide nanocomposite was characterised with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and X-ray diffractometry (XRD) to evaluate its morphology, crystallinity, composition and purity. An amino-modified single stranded DNA oligonucleotide probe synthesised based on complementary Coconut Cadang-Cadang Viroid (CCCVd) RNA sequence, was covalently bonded onto the surface of graphene/zinc oxide nanocomposite by the bio-linker 1-pyrenebutyric acid N-hydroxysuccinimide ester. The hybridisation events were monitored by electrochemical impedance spectroscopy (EIS). Under optimised sensing conditions, the single stranded CCCVd RNA oligonucleotide target could be quantified in a wide range of 1.0×10-11M to 1.0×10-6 with good linearity (R =0.9927), high sensitivity with low detection limit of 4.3×10-12M. Differential pulse voltammetry (DPV) was also performed for the estimation of nucleic acid density on the graphene/zinc oxide nanocomposite-modified sensing platform. The current work demonstrates an important advancement towards the development of a sensitive detection assay for various diseases involving RNA agents such as CCCVd in the future.
Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase the microbial yield for CAP patients. This study was designed to develop, optimize and evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP namely Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. Duplex and triplex real-time PCR assays were developed using five sets of primers and probes that were designed based on an appropriate specific gene for each of the above CAP pathogens. The performance of primers for each organism was tested using SYBR Green melt curve analysis following monoplex realtime PCR amplification. Monoplex real-time PCR assays were also used to optimize each primers-probe set before combining them in multiplex assays. Two multiplex real-time PCR assays were then optimized; duplex assay for the differential detection of S. pneumoniae and B. pseudomallei, and triplex assay for the atypical bacterial pathogens. Both duplex and triplex real-time PCR assays were tested for specificity by using DNA extracted from 26 related microorganisms and sensitivity by running serial dilutions of positive control DNAs. The developed multiplex real-time PCR assays shall be used later for directly identifying CAP causative agents in clinical samples.