scholarly journals PCR Primer Spesifik Berdasarkan Gen Cytochrome b untuk Deteksi Garangan (Herpestes javanicus) secara Molekuler

2020 ◽  
Vol 14 (1) ◽  
pp. 55
Author(s):  
Sena Adi Subrata ◽  
Subeno Subeno ◽  
Atus Syahbudin
Keyword(s):  
Data Collection ◽  
Cytochrome B ◽  
Dna Sequences ◽  
Organic Material ◽  
In Silico ◽  
Pcr Primers ◽  
Pest Species ◽  
Herpestes Javanicus ◽  
Pcr Primer

Garangan (Herpestes javanicus) merupakan salah satu spesies meso-carnivora yang berperan penting dalam ekosistem sebagai pemangsa. Kehadirannya dianggap mampu mengendalikan populasi spesies mangsa, termasuk beberapa spesies hama. Namun anggapan ini dianggap hanya anekdot karena kekurangan data ekologis pendukungnya sebagai dampak dari kesulitan pengamatan visual atas spesies ini. Penelitian ini bertujuan untuk merancang PCR primer untuk deteksi Garangan secara molekuler dari material organik yang ditinggalkan, misalnya kotoran. Perancangan PCR primer dimulai dengan memilih penanda spesifik Garangan yang melibatkan 51 sekuen DNA gen Cytochrome-b dari 19 spesies karnivora Jawa, menentukan primer forward dan reverse, dan menguji in-silico dan in vitro dari primer yang berhasil dirancang. Proses tersebut dilakukan dengan bantuan software MEGA 5 dan SP-Designer, dan memanfaatkan basis data genetik dari GenBank (NCBI). Uji in silico dan in vitro menunjukkan bahwa sekuen primer forward 5’- CAAATCACACCCACTCATTAAAATC-3’ dan reverse 5’-TGTGGGTTACTGATGAAAAGG-3’ akan mampu mendeteksi Garangan secara molekuler dari material organik spesies ini. Penelitian ini berkontribusi dalam pengumpulan data dasar kehadiran Garangan untuk mengumpulkan informasi lanjut tentang okupansi, distribusi spesies, dan pakannya. Informasi ini merupakan fondasi untuk memahami peran spesies ini dalam ekosistem. Specific PCR Primers Based on Cytochrome-b Gene for Molecular Detection of JavanMongoose (Herpestes javanicus)AbstractJavan Mongoose is a meso-carnivore species that have an important role in ecosystem as a predator. It is believed that its occurrence controls prey populations including some pest species. However, the belief is an anecdote because of lacking supportive data. Difficulty in visually observing the species is a major problem preventing data collection. This study aims to design PCR primer for detecting the Mongoose molecularly from organic material remaining, such as feces. The design starts with selecting specific marker from 51 DNA sequences of cytochrome-b. The DNA sequences were of 19 Javan carnivore collected from GenBank (NCBI). PCR primers were designed and tested using both in-silico and in-vitro techniques. The sequence collection and selection, and primer design process employed MEGA 5 and SP-Designer software. We successfully designed PCR primers: forward 5’- CAAATCACACCCACTCATTAAAATC-3’ dan reverse 5’-TGTGGGTTACTGATGAAAAGG-3’. The primer is capable of detecting Javan Mongoose from remaining organic material. The study contributed to basic data collection of Javan Mongoose for advanced studies such as occupancy modeling, species distribution modeling and diet analysis. This information is fundamental for understanding the role of the Javan Mongoose in an ecosystem.

scholarly journals New PCR Primer Pairs Specific for Cryptococcus neoformans Serotype A or B Prepared on the Basis of Random Amplified Polymorphic DNA Fingerprint Pattern Analyses

1999 ◽  
Vol 37 (2) ◽  
pp. 315-320 ◽  
Author(s):  
Francisco Hideo Aoki ◽  
Tamae Imai ◽  
Reiko Tanaka ◽  
Yuzuru Mikami ◽  
Hideaki Taguchi ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Primers ◽  
Dna Fingerprint ◽  
Serotype A ◽  
Specific Pcr ◽  
Fingerprint Pattern ◽  
Serotype B ◽  
Pcr Primer

Thirty-three strains of Cryptococcus neoformans were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for C. neoformans serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respectiveC. neoformans serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from C. neoformans serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical C. neoformans isolates is discussed.

scholarly journals Revisiting the Specificity of PCR Primers for Diagnostics of Xanthomonas citri pv. citri by Experimental and In Silico Analyses

Plant Disease ◽  
2013 ◽  
Vol 97 (3) ◽  
pp. 373-378 ◽  
Author(s):  
Suzy Delcourt ◽  
Christian Vernière ◽  
Claudine Boyer ◽  
Olivier Pruvost ◽  
Bruno Hostachy ◽  
...  
Keyword(s):  
Dna Sequences ◽  
In Silico ◽  
Large Strain ◽  
In Silico Analysis ◽  
Citrus Canker ◽  
Pcr Primers ◽  
Xanthomonas Citri ◽  
Canker Disease ◽  
Strain Collection ◽  
New Findings

Asiatic citrus canker disease, caused by Xanthomonas citri pv. citri, seriously impacts citrus production worldwide. Two pathogenic variants, A and A*/Aw, have been described within this pathovar. Two additional pathovars of X. citri with a limited geographic distribution and reduced pathogenicity, namely X. citri pvs. aurantifolii and bilvae, are also pathogenic to citrus and some rutaceous species. Rapid and reliable identification is required for these citrus pathogens, which are classified as a quarantine organism in citrus-producing countries. The specificity of nine polymerase chain reaction primers previously designed for the identification of X. citri pv. citri or citrus bacterial canker strains (both pvs. citri and aurantifolii) was assayed on a large strain collection (n = 87), including the two pathotypes of X. citri pv. citri, other genetic related or unrelated pathogenic xanthomonads, and saprophytic xanthomonads. This study gave congruent results with the original articles when testing the same strains or pathovars but the use of a broad inclusivity and exclusivity panel of strains highlighted new findings. Particularly, primers 2/3, 4/7, and KingF/R failed to provide amplification for three strains from the pathotype A*/Aw. Moreover, all pairs of primers detected at least one non-target strain. These data were supported by in silico analysis of the DNA sequences available from National Center for Biotechnology Information databases.

scholarly journals In silico Comparative Genomics of the Severe Acute Respiratory Syndrome Coronavirus 2 (COVID-19) and Designing of a Cost-effective RFLP-PCR Based Mass Screening Diagnostic Protocol

2020 ◽  
Author(s):  
Aditya Singh ◽  
Prateek Bhatia
Keyword(s):  
Comparative Genomics ◽  
Internal Control ◽  
In Silico ◽  
Working Hours ◽  
Cost Effective ◽  
Pcr Primers ◽  
Restriction Enzyme Digestion ◽  
Middle Income ◽  
The Status

SARS-CoV 2 also known as COVID-19 is a fast spreading coronavirus related disease that emerged from China in December 2019 and is currently attained the status of a pandemic. There are currently no drugs/ vaccines against the same and moreover limited diagnostic tests to identify the infection. Additionally, these tests are expensive and hence are exclusive for very highly suspected cases of the disease especially in developing countries. This is causing an under-diagnosis which is an alarming state of affairs, as even a single missed SARS-CoV 2 case would spread the disease exponentially and keep it in the community. Through this entirely in silico study, we have developed a cheaper and faster diagnostic method based on simple PCR and restriction enzyme digestion, commonly used in restriction fragment length polymorphism (RFLP) tests. Through comparative genomics, we found the closest neighbours of SARS-CoV 2 then found the highly conserved regions of the genome which were absent in SARS-CoV 1, its closest neighbour. Then we found restriction sites for various enzymes followed by designing of PCR primers flanking those sites. We have found the primer pair to produce a 401 bp amplicon and when digested by SwaI enzyme, it produces two fragments of lengths 216 bp and 185 bp. As an internal control, GAPDH primers are pooled with the SARS-CoV 2 primers as the patient sample will also include human RNA mixed with the viral RNA. This primer pair gives an amplicon of 131 bp and hence a negative sample should show a single band of 131 bp while a positive digested sample will give three bands of 401 bp, 216 bp and 131 bp. The primers are specific to SARS-CoV 2 only and can additionally be used for SYBR green based real time quantification of viral load. The developed tests have not yet been tested in vitro due to stressed out working hours in the only pathogenic virus handling laboratory in our institute. Nonetheless, this study works as a head start for other laboratories to rapidly test the suggested protocols in vitro and make available a cheaper alternative test for SARS-CoV 2 which would especially be beneficial for the lower to middle income countries.

scholarly journals Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

2020 ◽  
Author(s):  
Daniel C. F. Lanza ◽  
João P. M. S. Lima ◽  
Selma M. B. Jerônimo
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
In Silico ◽  
False Negative ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Multiple Sequence ◽  
Conserved Regions ◽  
Polymerase Chain

Abstract Accurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). Those primers and probes were subsequently validated in 3067 SARS-CoV-2 whole-genome sequences. From these analyses, we obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, or apicomplexan sequences. The availability of these primer and probe sequences will make it possible to accelerate the beginning of in vitro tests in order to validate more efficient protocols for the identification of SARS-CoV-2.

IN VITRO/IN SILICO CHARACTERIZATION OF ACTIVE AND PASSIVE STRESSES IN CARDIAC MUSCLE

2012 ◽  
Vol 10 (2) ◽  
pp. 171-188 ◽  
Author(s):  
Markus Boel ◽  
Oscar J. Abilez ◽  
Ahmed N Assar ◽  
Christopher K. Zarins ◽  
Ellen Kuhl
Keyword(s):  
Cardiac Muscle ◽  
In Silico ◽  
In Silico Characterization

Role of 4-O Galloylchlorogenic Acid in Lung Cancer- An Insilico Approach

2017 ◽  
Vol 9 (5) ◽  
Author(s):  
Jaynthy C. ◽  
N. Premjanu ◽  
Abhinav Srivastava
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
In Silico ◽  
Computational Study ◽  
Regulation Mechanism ◽  
Down Regulation ◽  
Fruit Extract ◽  
In Vitro Techniques ◽  
Discovery Studio

Cancer is a major disease with millions of patients diagnosed each year with high mortality around the world. Various studies are still going on to study the further mechanisms and pathways of the cancer cell proliferation. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. In cancer development increased core fucosylation by FUT8 play an important role in cell proliferation. Down regulation of FUT8 expression may help cure lung cancer. Therefore the computational study based on the down regulation mechanism of FUT8 was mechanised. Sapota fruit extract, containing 4-Ogalloylchlorogenic acid was used as the inhibitor against FUT-8 as target and docking was performed using in-silico tool, Accelrys Discovery Studio. There were several conformations of the docked result, and conformation 1 showed 80% dock score between the ligand and the target. Further the amino acids of the inhibitor involved in docking were studied using another tool, Ligplot. Thus, in-silico analysis based on drug designing parameters shows that the fruit extract can be studied further using in-vitro techniques to know its pharmacokinetics.

Discovery of New AKT1 Inhibitors by Combination of In silico Structure Based Virtual Screening Approaches and Biological Evaluations

2019 ◽  
Author(s):  
Filip Fratev ◽  
Denisse A. Gutierrez ◽  
Renato J. Aguilera ◽  
suman sirimulla
Keyword(s):  
Virtual Screening ◽  
Success Rate ◽  
Protein Interactions ◽  
In Silico ◽  
Biological Data ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Screening Protocol ◽  
Screening Approaches

AKT1 is emerging as a useful target for treating cancer. Herein, we discovered a new set of ligands that inhibit the AKT1, as shown by in vitro binding and cell line studies, using a newly designed virtual screening protocol that combines structure-based pharmacophore and docking screens. Taking together with the biological data, the combination of structure based pharamcophore and docking methods demonstrated reasonable success rate in identifying new inhibitors (60-70%) proving the success of aforementioned approach. A detail analysis of the ligand-protein interactions was performed explaining observed activities.<br>

Developing FGFR4 Inhibitors As Potential Anti-Cancer Agents Via In Silico Design, Supported by In Vitro and Cell-Based Testing

2013 ◽  
Vol 999 (999) ◽  
pp. 1-15
Author(s):  
H.K. Ho ◽  
G. Nemeth ◽  
Y.R. Ng ◽  
E. Pang ◽  
C. Szantai-Kis ◽  
...  
Keyword(s):  
In Silico ◽  
In Silico Design ◽  
Anti Cancer
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