Development of Tetra-primer Amplification Refractory Mutation System (ARMS) PCR for Detection of CHRNA3 rs8040868

BACKGROUND: Single nucleotide variations (SNV) have been mapped to be associated with several human conditions and diseases. To validate the association between SNV to certain human traits or diseases, a large number of subjects must be included. Thus, in need of a fast, relatively economic, and reliable genotyping method. This can be achieved through the use of tetra-primer amplification refractory mutation system polymerase chain reaction (Tetra-primer ARMS PCR). This study reports strategy to develop Tetra-primer ARMS PCR-based genotyping of CHRNA3 rs8040868.METHODS: The optimization of Tetra-primer ARMS PCR was done through these steps: identification of gene sequence and position of single mutation; designing outer and inner PCR primers; amplification of target gene fragments through PCR by using outer primer; confirming genotype of the PCR product by using sequencing; determining an optimum ratio of outer and inner primer; and determining optimum annealing temperature and cycles for the PCR program. The PCR products were run in 2% gel agarose electrophoresis and visualized under UV illumination.RESULTS: Outer and inner primer ratio of 1:3 with annealing temperature of 64.4°C and 40x cycles was found to be the most optimum condition. Tetra-primer ARMS PCR was able to confirm the results of the DNA sequence of 2 samples, confirming wild-type variants (TT allele) and the heterozygous mutant (CT allele).CONCLUSION: Tetra-primer ARMS PCR was able to genotype rs8040868 of the CHRNA3 gene.KEYWORDS: tetra-primer ARMS PCR, CHRNA3, rs8040868, genotyping

Introducing Electrospray as a Potent Technique to Deliver Chitosan/pDNA Nanoparticles to Eukaryotic Cells

Electrospray technique has received increasing attentions for intracellular gene delivery as well as production of nanoparticles. In this study, chitosan/pDNA nanoparticles with N/P ratio of 5 were prepared and transferred to HEK293T cells by electrospray technique. Physicochemical characterization of prepared nanoparticles, including size, zeta potential and entrapment efficiency was performed and attachment of pDNA to chitosan was confirmed by gel agarose electrophoresis. Moreover, transfection efficiency was investigated using flow cytometry. MTT assay was performed for cell viability studies. Nanoparticles were prepared at three pDNA concentrations of 10, 55 and 100 μg/ml in fixed N/P ratio. Size of nanoparticles was obtained as 110, 188 and 240 nm, using DLS. SEM showed size of 102.34 ± 10.66 nm for samples having 55 μg/ml pDNA. Zeta potential and entrapment efficiency were +25 mv and 85±4%m respectively. The effect of pDNA concentration, electrospray time and incubation time on transfection efficiency was investigated using Box-Behnken design. Percent of GFP-positive cells was 41.05 ± 3.04% which was taken as an indicator of transfection efficiency. Transfection efficiency of this method was then compared with that of calcium phosphate (31.1 ± 2.4%), showing improved efficiency. Considering the fact that electrospray is an easy, low cost, one-step process which makes low damage to cells and produces monodispersed nanoparticles, the method is introduced as a fascinating approach in gene transfection.

Detection of B1 gene as Toxoplasmosis marker in women of childbearing age in West Bandung Regency, Indonesia

Congenital toxoplasmosis can cause damage and death to the fetus, to prevent this case, toxoplasmosis testing is important for the woman of childbearing age. One of the methods to screening the presence of T. gondii in the blood is Polymerase Chain Reaction (PCR). One of the T. gondii genes which can be used as a marker is the B1 gene. There are many toxoplasmosis cases in Indonesia, but the data is still difficult to find in West Bandung Regency. This study aimed to determine the number of toxoplasmosis cases in a woman of childbearing age in West Bandung Regency using the B1 gene as a marker and to determine the factors that influence these cases by conducting statistical analysis on the results of the questionnaire. The sample used in this study was 50 women of childbearing age (got married and domiciled in West Bandung). All samples have met the inclusion criteria and signed the informed consent. DNA from blood specimens was isolated using the Wizard Genomic DNA Purification Kit. The concentration and purity of isolated DNA were measured using a nanodrop device. Besides, the B1 gene from T. gondii was amplified using a pair of specific primers and visualized by the agarose electrophoresis method. Data were analyzed using the logistic regression method. The results showed that 7 women of childbearing age women (14%) in West Bandung Regency had toxoplasmosis. Frequent contact with pets, especially cats, was a significant factor (p <0.005) in this disease transmission.

Induced Disassembly of a Virus-Like Particle under Physiological Conditions for Venom Peptide Delivery

ABSTRACTVirus-like nanoparticles (VLPs) show considerable promise for the in vivo delivery of therapeutic compounds such as bioactive venom peptides. While loading and targeting protocols have been developed for numerous VLP prototypes, induced disassembly under physiological conditions of neutral pH, moderate temperature, and aqueous medium, remain a challenge. Here, we implement and evaluate a ring-opening metathesis polymerization (ROMP) general mechanism for controllable VLP disassembly that is independent of cell-specific factors or the manipulation environmental conditions such as pH and temperature that cannot be readily controlled in vivo. The ROMP substrate norbornene is covalently conjugated to surface-exposed lysine residues of a P22 bacteriophage-derived VLP, and ROMP is induced by treatment of water-soluble ruthenium catalyst AquaMet. Disruption of the P22 shell and release of a GFP reporter is confirmed via native agarose electrophoresis and quantitative microscopy and light scattering analyses. Our ROMP disassembly strategy does not depend on the particular structure or morphology of the P22 nanocontainer and is adaptable to other VLP prototypes for the potential delivery of venom peptides for pharmacological applications.Abstract Figure

Estimation of bacterial number in the water, sediment and biofilm of Badek and Mewek River (Malang, Indonesia) using Plate Count and eDNA Method

Ulinuha D, Andayani S, Hertika AMS, Kilawati Y. 2020. Estimation of bacterial number in the water, sediment, and biofilm of Badek and Mewek River (Malang, Indonesia) using Plate Count and eDNA Method. Biodiversitas 21: 3832-3836. Several methods have been developed to quantify bacterial numbers in the environment, but the studies is still continued to provide the most effective and efficient method. This study was aimed to compare the capacities of two different methods (Plate Count and eDNA Method) to estimate the bacterial number in the water, sediment, and biofilm of Badek and Mewek River (Malang-Indonesia). The Plate Count Method was carried out on a Plate Count Agar (PCA) which was incubated for 24 hours at 37oC. The eDNA method was performed by slow agitation (1,800 rpm) to extract the environmental DNA of bacteria. Quantification of bacteria was obtained from the eDNA band intensity after electrophoresis in 1% gel agarose electrophoresis. Smartladder was used as a marker for the eDNA Method. The result showed that the eDNA Method was efficient for the estimation of bacterial numbers in the sediment and biofilm of Badek and Mewek River. However, this method was not suitable for estimating bacterial numbers in the water.

Serum Protein Gel Agarose Electrophoresis in Captive Tigers

Given the endangered status of tigers (Panthera tigris), the health of each individual is important and any data on blood chemistry values can provide valuable information alongside the assessment of physical condition. The nature of tigers in the wild makes it is extremely difficult to obtain biological samples from free-living subjects, therefore the values obtained from captive tigers provide very useful data. Serum protein electrophoresis is a useful tool in the diagnosis and monitoring of a number of diseases. In this study, we evaluated agarose gel serum protein electrophoresis on samples from 11 healthy captive tigers. Serum electrophoresis on all 11 tiger samples successfully separated proteins into albumin, α1, α2, β1, β2 and γ globulin fractions as in other mammals. Electrophoretic patterns were comparable in all tigers. Mean± standard deviation or median and range values obtained for each protein fraction in healthy tigers were, respectively: 3.6 ± 0.2, 0.21 (0.2–0.23), 1.2 ± 0.2, 10.7 ± 0.2, 0.4 (0.3–0.6), 1.2 (1–1.8) gr/dL. The results of this preliminary study provide the first data on serum electrophoretic patterns in tigers and may be a useful diagnostic tool in the health assessment of this endangered species.

Associated factors in distinguishing patients with brucellosis from suspected cases

Background To investigate the risk factors for brucellosis in suspected cases of the disease. Methods A self-designed questionnaire was developed to collect data from 3557 people whose initial visit site was the Songyuan Center for Disease Control and Prevention (CDC) from January 1st, 2009 to December 31st, 2012. After collecting blood samples, a plate agglutination test (PAT) and serum agglutination test (SAT) were used to distinguish the patients with brucellosis from the suspected cases. Results Sex, occupation (farmers and herdsmen), contact with abortion products, and contact with feces were the main risk factors for brucellosis in the suspected cases (all P < 0.05). No difference existed between the confirmed cases and suspected cases in the demographic characteristics, contact with animals (except swine), contact with substances, or clinical symptoms (except fever). However, the confirmed cases showed significant differences from people without brucellosis in demographic characteristics, contact with animals (except cattle and swine), contact with substances, and clinical symptoms. Suspected cases exhibited significant differences from people without brucellosis in the demographic characteristics (except education), contact with animals (except swine), contact with substances (except dust), and clinical symptoms (except chills and acratia). Brucella was cultured from the blood samples of three of 30 suspected cases with fever. Using AMOS-PCR and agarose electrophoresis, the detailed species of Brucella strain was identified as Brucella melitensis. Conclusions Abortion products and feces are the main risk factors for brucellosis in suspected cases of the disease. Pyrexia in suspected cases with a history of contact with abortion products or feces should raise suspicion for the disease.

Deteksi Gen sHBsAg sebagai Penanda Koinfeksi Hepatitis B pada Pengidap HIV

<p>Bandung is the city with the highest number of Human Immunodeficiency Virus (HIV) sufferers in West Java. HIV patients often have co-infection with Hepatitis B Virus (HBV). This condition can increase the risk of cirrhosis, liver cancer, and death. Co-infection status can be detected by the presence of the HBsAg gene amplified using the nested Polymerase Chain Reaction (nested PCR) method. This study aimed to decide the number of HBV co-infection cases with the sHBsAg gene as a marker and factor influencing the presence of the gene in HIV patients in Bandung. This research used 50 human samples domiciled in Bandung which have been infected with HIV and never had a hepatitis test. Taking blood specimens was done on the people who had signed the informed consent. The detection of the sHBsAg gene started with genomic DNA isolation. Moreover, the purity and concentration of DNA isolation results were measured by the Nanodrop. The amplification process of the sHBsAg gene was done twice using two pairs of the specific primary. The amplification results were visualized by agarose electrophoresis method. Then, the data collected was analyzed by a logistic regression method. The laboratory results showed that 18 people (36%) had HIV-HBV co-infection, marked by the presence of the sHBsAg gene in their blood. Unsafe sexual activity, syringe drug use, and a vaccination profile were factors that significantly influence (p&lt;0.05) on this co-infection.</p>