Single Strand Conformation Polymorphism Method for Initial Detection DNA Sequences Homogeneity

Abstract: In molecular phylogenetic study, homogeneity of DNA sequences is a prerequisite before putting it into practice. Internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) has been common in phylogenetic study, but a homogenous sequence is often difficult to obtain. Here we use single-stranded conformation polymorphism (SSCP) method to detect homogeneity for nine pooled amplified products of ITS region. Our results suggested that SSCP method has been applicable in detection homogeneity of ITS region prior to using it in sequencing processes.

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Control of Fusarium Wilt of Chili With Chitinolytic Bacteria

Abstract: Biological control of plant disease using antagonistic microorganism has been obtaining much attention and implemented for decades. One of the potential microorganisms used in this strategy is chitinolytic bacteria. Utilization of this bacteria ranges from cell life, enzymes, genes, or other metabolites. In this study, we examined the ability of chitinolytic bacteria as a biocontrol agent of Fusarium wilt of red chili (Capsicum annuum L.) seedlings. The ability of chitinolytic bacteria to suppress the disease was evaluated by soaking red chili seeds in the bacterial isolates solution for 30 minutes prior seedling. Percentage of seedling of treated chili seed at end of study (4-weeks) ranging from 46 to 82.14%. Relative reduction of the seedling damping-off was observed in all bacterial treatment ranged from 28.57 to 60.71%. Furthermore, manifestation of bacterial suppression to Fusarium wilt was also exhibited by increasing of seedling height (ranged from 7.33 to 7.87 cm compared to 6.88 cm) and dry-weight (ranged from 2.7 to 4.3 mg compared to 2.3 mg). However, no significant effect was observed in leaf number. Then, from all chitinolytic isolates tested, BK08 was the most potential candidate for biological control agent of Fusarium wilt in chili seedling.

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Troubleshooting In Expression And Purification Of Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus Nucleocapsid Protein In Escherichia Coli Bl21

Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, we reported effects of several conditions on purity and homogeneity of recombinant SARS-CoV N protein expressed in E. coli BL21. The SARS-CoV N gene was reverse transcribed and amplified by the reverse transcription-polymerase chain reaction (RT-PCR) technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the target gene into pQE-80L. After inserting the recombinant plasmid (pQE80-N) into E. coli, the recombinant protein (6 x His tag-N protein fusion) was expressed by inducing the bacterial cells with 0.1-0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) for 1-5 h. The protein recombinant were extracted from the bacterial cells by NTT buffer containing 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction of E. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mM imidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.

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Detection Of Human Group A And C Rotaviruses In Pediatric Patients With Acute Gastroenteritis By Real Time RT-PCR Assay: A Preliminary Study

Rotavirus causes 25–55% of all hospital admissions for diarrhea and approximately 611.000 deaths every year in developing countries. Clinically, it is not possible to recognize the diarrhea caused by rotavirus and other infections. To know a causative agent of rotavirus gastroenteritis, availability of an accurate diagnosis assay is necessary. Therefore, we developed real time RT-PCR assay (rRT-PCR) assay for confirmation of infections of Group A or C rotaviruses simultaneously. A total of 54 stool samples obtained from pediatric patients (< 5 years old) was used in this study. All samples were tested for Group A rotavirus by Serological rapid test. Result of serological rapid test was compared with rRT-PCR assay to obtain the test accuracies of both assays. Result of this study showed that rates of positive testing for Group A rotavirus by serological rapid test and the rRT-PCR assay were 22.22% and 18.50%, respectively. Forty-two serology-negative specimens for Group A rotavirus were also PCR negative (100% specificity). Two serology-positive specimens for Group A rotavirus was rRT-PCR negative (confirmed by electrophoresis gel); therefore, rRT-PCR assay represents the decrease of 3.70% in the number of specimens that are positive for Group A rotavirus. For Group C rotavirus, all tested samples were no rRT-PCR positive and the results need to be confirmed in the future.

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Types and Functions of The Microscope

Compound Light Microscopy: functions to see small objects with bright field
Darkfield Microscope: observed microbial can not be seen with ordinary light microscope, no can be characterized by standard methods, or experience distortion after staining. This microscope condenser using dark field condenser is not allow the light transmitted through the specimen continue into the objective lens.
Phase-Contrast Microscopy: allowing examination in detail the internal structure of microbial life. Using a special condenser.
Differential Interference Contrast Microscopy: This microscope allows viewing of objects into 3 dimension. The light is broken down and recombined with special prism.
Fluorescence microscopy: using fluorescent materials thus giving the color, use a uv light or near uv. If the object is not naturally fluorescent, objects can be colored with a fluorescent material called a fluorochrome. Examples of auramine O to give yellow color with UV, so absorbed with Mycobacterium tuberculosis; fluorescein isothiocyanate (FITC) gives bright yellow color, is absorbed by Bacillus anthracis. Specialized techniques in microscopy is a technique called antibody-fluorescent or immunofluoresen, using antigen-antibody reaction.
Electron Microscopy: there are two types of transmission electron microscopy (TEM) and scanning electron microcopy (SEM). The difference between the two lies in the use. TEM is used to view the preparations ultra-thin slices, preparations can be colored. In the SEM specimens do not need to be sliced ​​thin with results 3-dimensional observations. Both the microscope can enlarge the shadow of the object of observation until tens of thousands of times, something that can not be done with ordinary light microscope. Lens on the microscope This form of the electric field.

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