Ex vivo Study of Antimalarial Activity of Canarium Odontophyllum Leaf Extracts Against Plasmodium Berghei NK65

Background: Malaria is a parasite that is transmitted to human through the bite of a female Anopheles mosquito. Every year human was exposed to the threat of malaria infection. This disease becomes more fatal as these parasites show resistance towards the drug available. Thus, searches for new antimalarial drug are crucial. This study was carried out to evaluate the antimalarial activity in Canarium odontophyllum leaf extracts (methanol, acetone and aqueous) against erythrocytes infected with Plasmodium berghei NK65 using Plasmodium Lactate Dehydrogenase (pLDH) Assay and SYBR green I fluorescence Assay. Method: Three types of solvents were used to extracts Canarium odontophyllum leaf according to increasing polarity index; acetone, methanol and aqueous. These extracts were made into eight-fold serial dilution; concentrations ranging from 0.00001μg / ml as the lowest concentration until 100 μg / ml as the highest concentration and further tested on Plasmodium berghei NK65 infected erythrocytes via ex-vivo. The IC50 (inhibition concentration) 50 readings were taken at the point of 5% parasitemia level and in the synchronization process. Both PLDH assay and SYBR green I fluorescence assay were being carried out simultaneously. Result: The One-way ANOVA showed that there is no significant difference between extracts at 5% parasitemia level, even so methanol was further tested on synchronization process as it showed the lowest reading of IC50 among the three extracts for PLDH assay and SYBR green I fluorescence assay respectively, (IC50 0.00045μg / ml, 0.002 μg / ml). For synchronization stages, the One-way ANOVA result showed there is no significant difference between stages of morphology. However, methanol extracts showed the most potent on schizont, (1.16x10-5 μg / ml ) and young trophozoite, (0.00195 μg / ml ) stages for each method respectively. Conclusions: All three extracts of Canarium odontophyllum leaf were effective on Plasmodium berghei NK65, however methanol showed most promising results and further research on the fractions were required for proper drug development. The Ministry of Higher Learning funded this project, Government of Malaysia, under the Fundamental Research Grant Scheme Code No. FRGS/2/2014/SG05/UKM/02/3.

A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

Highly Sensitive Detection for Mercury Ions Using Graphene Oxide (GO) Sensors

The mercury ion (Hg2+) is one of the heavy metal ions, and its presence in trace amounts can cause physiological damage to an organism. Traditional methods of Hg2+ detection have been useful but have also had numerous limitations and challenges, and as a result, it is important to design new and sophisticated methods that can aid in the detection of Hg2+. In this paper, two fluorescent dyes, carboxyfluorescein (FAM) and SYBR Green I, were used to label and intercalate DNA probes immobilized on the surface of graphene oxide (GO) for sensors to detect Hg2+. FAM and SYBR Green I dye share close excitation and emission wavelength spectra, which can promote and amplify the detection of signals, and also increase the limit of detection (LOD). The results showed that the limit of detection in this method was 0.53 nM. Moreover, when the sensors with double amino groups on the surface of GO were carried out to detect Hg2+, a limit of detection was improved to 0.43 nM. The sensors were then applied in the real sample. The results show that this method has a promising potential in Hg2+ detection.

Ultra-Rapid Drug Susceptibility Testing for Klebsiella pneumoniae Clinical Isolates in 60 Min by SYBR Green I/Propidium Iodide Viability Assay

BackgroundWe aimed to optimize and validate the drug susceptibility test (DST) assay by SYBR Green I/PI (SG-PI) method using a panel of 89 Klebsiella pneumoniae clinical isolates in comparison with the conventional DST method to three most important antibiotics used for treatment of this bacterial infection, including imipenem, cefmetazole, and gentamicin.MethodsBy staining with SYBR Green I and PI dyes, green fluorescence and red fluorescence, which linearly correlated with the percentages of live and dead or membrane damaged cells, respectively, were used to produce two standard curves to calculate the relative cell membrane impermeable rates for each log and stationary phase cultures. Stationary phase K. pneumoniae cells were used in imipenem and cefmetazole SG-PI DST assay whereas log phase cells were used in the gentamicin assay. The conventional broth microdilution method was used as a gold standard for DST for comparison.ResultsData showed that after antibiotic treatment for 30–60 min, the antibiotic-resistant K. pneumoniae strains had significantly higher numbers of surviving cells than the susceptible strains at different concentrations of imipenem, cefmetazole, and gentamicin, where the average relative membrane impermeable rates were 88.5, 92.5, and 103.8% for resistant clinical strains, respectively, and 9.1, 49.3, and 71.5% for susceptible strains, respectively. Overall, the total concordances between the ultra-rapid SG-PI method and conventional minimal inhibitory concentration assay in diagnosing imipenem, cefmetazole and gentamicin resistance were high and were 96.6% (86/89), 95.4% (83/87), and 95.5% (85/89), respectively.ConclusionWe demonstrate that our novel SG-PI assay can accurately and stably detect resistance to different antibiotics in clinical isolates of K. pneumoniae in an ultra-fast manner in 60–90 min.

Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection

Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the rpoB gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuberculosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings.