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Choice of PCR microtube can impact on the success of long-range PCRs

Chua EW, Miller AL, Kennedy MA

Anal Biochem, 2015 May 15;477:115-7.
PMID: 25766577 DOI: 10.1016/j.ab.2015.02.023

We compared four brands of microtubes with respect to their suitability for long-range polymerase chain reactions (PCRs). One of the four brands was found to have an inhibitory effect, decreasing PCR yields. The effect was universal across different PCR or enzyme systems. Increased ultraviolet absorbance suggests leaching of unknown chemical species into PCR mixtures. However, this could not be confirmed by high-performance liquid chromatography-mass spectrometry analysis. Nevertheless, our article demonstrates a clear impact of the choice of microtubes on long-range PCR success. Due consideration should be given to the PCR microtubes when determining optimal reaction conditions for long-range PCR.

Matched MeSH terms: Polymerase Chain Reaction/instrumentation*
Quantitative RT PCR approach to evaluate the neurogenic and gliagenic gene expression of cultured human amniotic epithelial cells

Tan GC, Simat SF, Abdul Rahman H, Tan AE, Chua KH

Med J Malaysia, 2008 Jul;63 Suppl A:51-2.
PMID: 19024979

The aim of the study is to determine the neuronal and glial gene expression of cultured human amniotic epithelial cells (HAECs) in serial passages. HAECs obtained from human term placentae were cultured in F12:DMEM (1:1) + 10% FBS +10ng/ml EGF in serial passages. Quantitative RT-PCR was used to assess the gene expression analysis. The results showed that the cultured HAECs expressed the neural stem cell genes (Nestin, NSE and Vimentin), mature neuronal genes (TH, MAP-2, beta-III-tubulin and NFM) and glial genes (CNPase, MBP and Olig). These neural stem cell genes increased with serial passages while the genes expression for mature neuronal and glial cells were downregulated. These results suggested that HAECs may promote or involve in neurogenesis and gliagenesis.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/instrumentation
Neuro-genetic optimization of temperature control for a continuous flow polymerase chain reaction microdevice

Lee HW, Arunasalam P, Laratta WP, Seetharamu KN, Azid IA

J Biomech Eng, 2007 Aug;129(4):540-7.
PMID: 17655475

In this study, a hybridized neuro-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA), is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated reaction zones of 94 degrees C, 65 degrees C, and 72 degrees C for the denaturing, annealing, and extension processes, respectively, to complete a cycle of polymerase chain reaction. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of +/-1 degree C or better, irrespective of changing ambient conditions. Results obtained from the FEA simulation shows good comparison with published experimental work for the temperature control in each reaction zone of the microfluidic channels. The simulation data are then used to train the ANN to predict the temperature distribution of the microfluidic channel for various heater input power and fluid flow rate. Once trained, the ANN analysis is able to predict the temperature distribution in the microchannel in less than 20 min, whereas the FEA simulation takes approximately 7 h to do so. The final optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. Finally, the GA optimized results are used to build a new FEA model for numerical simulation analysis. The simulation results for the neuro-genetic optimized CPCR model and the initial CPCR model are then compared. The neuro-genetic optimized model shows a significant improvement from the initial model, establishing the optimization method's superiority.

Matched MeSH terms: Polymerase Chain Reaction/instrumentation
Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain

Hossain MAM, Ali ME, Sultana S, Asing, Bonny SQ, Kader MA, et al.

J Agric Food Chem, 2017 May 17;65(19):3975-3985.
PMID: 28481513 DOI: 10.1021/acs.jafc.7b00730

Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/instrumentation
Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules

Mohamad NA, Mustafa S, Khairil Mokhtar NF, El Sheikha AF

J Sci Food Agric, 2018 Sep;98(12):4570-4577.
PMID: 29505123 DOI: 10.1002/jsfa.8985

BACKGROUND: The pharmaceutical industry has boosted gelatin consumption worldwide. This is supported by the availability of cost-effective gelatin production from porcine by-products. However, cross-contamination of gelatin materials, where porcine gelatin was unintentionally included in the other animal sources of gelatin, has caused significant concerns about halal authenticity. The real-time polymerase chain reaction (PCR) has enabled a highly specific and sensitive animal species detection method in various food products. Hence, such a technique was employed in the present study to detect and quantify porcine DNA in gelatin using a molecular beacon probe, with differences in performance between mitochondrial (cytochrome b gene) and chromosomal DNA-(MPRE42 repetitive element) based porcine-specific PCR assays being compared.
RESULTS: A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA.
CONCLUSION: The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/instrumentation
Fulltext Droplet digital polymerase chain reaction (ddPCR) for the detection of Plasmodium knowlesi and Plasmodium vivax

Mahendran P, Liew JWK, Amir A, Ching XT, Lau YL

Malar J, 2020 Jul 10;19(1):241.
PMID: 32650774 DOI: 10.1186/s12936-020-03314-5

BACKGROUND: Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease transmission in Malaysia. The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of P. knowlesi and P. vivax, and compare this method to nested PCR and qPCR.
METHODS: The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.
RESULTS: The duplex ddPCR assay had higher analytical sensitivity (P. vivax = 10 copies/µL and P. knowlesi = 0.01 copies/µL) compared to qPCR (P. vivax = 100 copies/µL and P. knowlesi = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity (P. vivax = 80% and P. knowlesi = 90%) and clinical specificity (P. vivax = 87.84% and P. knowlesi = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.
CONCLUSIONS: Overall, the ddPCR assay demonstrated acceptable efficiency in detection of P. knowlesi and P. vivax, and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay's clinical sensitivity and specificity.

Matched MeSH terms: Polymerase Chain Reaction/instrumentation
Rapid detection of porcine DNA in processed food samples using a streamlined DNA extraction method combined with the SYBR Green real-time PCR assay

Tan LL, Ahmed SA, Ng SK, Citartan M, Raabe CA, Rozhdestvensky TS, et al.

Food Chem, 2020 Mar 30;309:125654.
PMID: 31678669 DOI: 10.1016/j.foodchem.2019.125654

A specialized DNA extraction method and a SYBR Green quantitative polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and incorporated internal controls. We improved the genomic DNA extraction method, and reduced extraction times to the minimum. The method was validated for specificity, sensitivity (0.001% w/w) and robustness, and values were compared with those of a commercially available kit. We also tested our method using 121 processed food products and consistently detected amplification only in samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a valuable new method for detecting food adulteration with pork that is superior to existing quality control approaches.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/instrumentation
STR data for the AmpFlSTR Profiler loci from the three main ethnic population groups (Malay, Chinese and Indian) in Malaysia

Lim KB, Jeevan NH, Jaya P, Othman MI, Lee YH

Forensic Sci Int, 2001 Jun 01;119(1):109-12.
PMID: 11348801

Allele frequencies for the nine STRs genetic loci included in the AmpFlSTR Profiler kit were obtained from samples of unrelated individuals comprising 139-156 Malays, 149-153 Chinese and 132-135 Indians, residing in Malaysia.

Matched MeSH terms: Polymerase Chain Reaction/instrumentation