Identifikasi molekuler rotifer Brachionus sp. asal perairan Tumpaan, Minahasa Selatan

2017 ◽  
Vol 5 (1) ◽  
pp. 56
Author(s):  
Jefri Sahari ◽  
Joice Rimper ◽  
Stenly Wullur
Keyword(s):  
Brachionus Plicatilis ◽  
Evolutionary Genetics ◽  
Read Length ◽  
Local Alignment ◽  
Chain Reaction ◽  
Query Cover ◽  
Rotifer Brachionus Plicatilis ◽  
Search Tool ◽  
Polymerase Chain ◽  
Dna Template

Rotifer yang digunakan dalam penelitian ini berasal dari Tumpaan, Minahasa Selatan dan telah dikultur massal selama beberapa generasi.  DNA genom rotifer diekstraksi mengikuti prosedur qiagen DNeasy Blood & Tissue kit; amplifikasi gen COI (Cytochrome oxidase sub unit 1) dilakukan dengan bantuan mesin PCR (Polymerase chain reaction) menggunakan primer universal (LCO1490 (forward) dan HCO2198 (reverse));dan dilanjutkan dengan pengurutan nukleotida produk PCR. Pengolahan data hasil sekuens dilakukan dengan menggunakan program ABsequens dan MEGA (Molecular Evolutionary Genetics Analysis) Identifikasi spesies dilakukan dengan menggunakan teknik BLAST (Basic Local Alignment Search Tool) di situs Genbank. Hasil amplifikasi gen COI menggunakan DNA template ekstrak DNA genom rotifer terobservasi adanya pita DNA pada posisi sekitar 700 bp.Kualitas hasil pengurutan nukeotida menggunakan produk PCR menunjukan nilai CRL (contignous read length) dan QV20+(quality value lebih besar dari 20) yang tinggi (>600 nukleotida).Hasil BLAST menunjukkan bahwa rotifer dalam penelitian ini merujuk pada rotifer Brachionus plicatilis complex spesies.Maximum dan total score, prosentase query cover dan prosentase identity masing-masing pada nilai1003-1116, 87-96% dan 96-97%.

scholarly journals Evaluasi Deteksi Berbasis PCR untuk Bakteri Bifidobacterium longum dalam Sampel Feses Bayi dari Kota Manado

2020 ◽  
Vol 20 (1) ◽  
pp. 18
Author(s):  
Beivy Jonathan Kolondam
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Detection Method ◽  
Bifidobacterium Longum ◽  
Local Alignment ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Search Tool ◽  
Polymerase Chain

Bifidobakteria merupakan mikroflora yang umum hidup dalam usus manusia sejak bayi. Peran Bifidobacterium longum yang positif sebagai salah satu bakteri yang menunjang kesehatan inangnya membuat bakteri ini menjadi objek studi yang menarik. Salah satu instrumen dalam penelitian adalah adalah metode deteksi bakteri B. longum yang berbasis PCR (Polymerase Chain Reaction) gen 16S rRNA. Dengan mempertimbangkan bahwa perancangan primer untuk deteksi ini sudah lebih dari 20 tahun, penelitian ini bertujuan mengevaluasi hasil deteksi melalui PCR terhadap B. longum dalam feses bayi. Akurasi hasil dilihat melalui sekuensing terhadap hasil PCR sampel yang terdeteksi positif. Dua sampel feses bayi di Manado yang diperiksa menunjukkan hasil positif dan produk PCR tersebut dilakukan sekuensing. Panjang DNA yang nyata dari hasil deteksi ini yaitu 829 bp dan bukan 831 bp. Sekuens DNA kedua sampel ini identik satu sama lain. Hasil BLAST (Basic Local Alignment Search Tool) mengonfirmasi kesamaan 100% (identik) dari kedua specimen dari Kota Manado dengan sekuens gen 16S rRNA specimen bakteri B. longum yang telah ada dalam GenBank.Kata-kata kunci: Bifidobacterium longum, Polymerase Chain Reaction, deteksi, feses, bayi. Evaluation of PCR-Based Detection for Bifidobacterium longum in Infant Fecal Samples from Manado City ABSTRACTBifidobacteria are common members of the gut microflora of humans since infant. The Bifidobacterium longum has positive roles and one of supportive bacteria to the host, which made interesting as a study object. One instrument in studying this bacterial species is the detection method based on PCR of 16S rRNA gene. In consideration of the design of primers for this detection method is already more than 20 years, this research aimed to evaluate the PCR-based detection of B. longum in infant feces. The accuracy of the method was evaluated from sequencing of DNA fragment from positive results. Two fecal samples in Manado City shown positive result were sent for sequencing. The actual length of DNA amplified by PCR was 829 bp, not 831 bp. The DNA sequence of both samples were identical to each other. The BLAST (Basic Local Alignment Search Tool) result confirmed the similarity of both samples from Manado with 16S rRNA gene sequence of B. longum specimens in GenBank.Keywords: Bifidobacterium longum, Polymerase Chain Reaction, detection, feces, infant.

scholarly journals KARAKTERISTIK SEKUEN cDNA PENGKODE GEN ANTI VIRUS DARI UDANG WINDU, Penaeus monodon

2016 ◽  
Vol 4 (1) ◽  
pp. 1
Author(s):  
Andi Parenrengi ◽  
Alimuddin Alimuddin ◽  
Sukenda Sukenda ◽  
Komar Sumantadinata ◽  
Andi Tenriulo
Keyword(s):  
Polymerase Chain Reaction ◽  
Penaeus Monodon ◽  
Viral Gene ◽  
Local Alignment ◽  
Chain Reaction ◽  
Crustacean Species ◽  
Viral Genes ◽  
Tiger Prawn ◽  
Search Tool ◽  
Polymerase Chain

Transgenesis pada ikan merupakan sebuah teknik modern yang berpotensi besar dalam menghasilkan organisme yang memiliki karakter lebih baik melalui rekombinan DNA gen target termasuk gen anti virus dalam peningkatan resistensi pada udang. Gen anti virus PmAV (Penaeus monodon Anti Viral gene) merupakan salah satu gen pengkode anti virus yang berasal dari spesies krustase. Penelitian ini dilakukan untuk mengetahui karakteristik gen anti virus yang diisolasi dari udang windu, Penaeus monodon. Isolasi gen anti virus menggunakan metode Polymerase Chain Reaction (PCR) dan selanjutnya dipurifikasi untuk sekuensing. Data yang dihasilkan dianalisis dengan program Genetyx Versi 7 dan basic local alignment search tool (BLAST). Hasil penelitian menunjukkan bahwa gen anti virus PmAV yang berhasil diisolasi dari cDNA udang windu dengan panjang sekuen 520 bp yang mengkodekan 170 asam amino. BLAST-N menunjukkan tingkat similaritas yang sangat tinggi (100%) dengan gen anti virus yang ada di GeneBank. Komposisi asam amino penyusun gen anti virus yang paling besar adalah serin (10,00%), sedangkan yang terkecil adalah asam amino prolin dan lisin masing-masing 1,76%. Analisis sekuen gen dan deduksi asam amino (BLAST-P) memperlihatkan adanya C-type lectin-like domain (CTLD) yang memiliki kemiripan dengan gen C-type lectin yang diisolasi dari beberapa spesies krustase.Transgenic fish technology is a potential modern technique in producing better character organism through DNA recombinant of target genes including anti viral gene for improvement of shrimp immunity. PmAV (Penaeus monodon Anti Viral) gene is one of anti viral genes isolated from crustacean species. The research was conducted to analyze the characteristics anti viral gene isolated from tiger prawn, Penaeus monodon. Anti viral gene was isolated using Polymerase Chain Reaction (PCR) technique and then purified for sequencing. Data obtained were analyzed using Genetyx Version 7 software and basic local alignment search tool (BLAST). The results showed that the PmAV antiviral gene has been isolated from cDNA of tiger prawn at the position of approximately 520 bp consisting of 170 amino acids. BLAST-N showed high similarity (100%) compared to the other anti viral genes deposited at the GeneBank. The highest percentage of amino acid encoding anti viral gene is serine (10.00%), while the lowest is proline and lysine (1.76%). Sequence analysis and amino acid deduction (BLAST-P) revealed a C-type lectin-like domain (CTLD) that is similar with the C-type lectin gene isolated from several crustacean species.

Kato–Katz thick smears as a DNA source of soil-transmitted helminths

2018 ◽  
Vol 94 ◽  
Author(s):  
K.J.L. Monteiro ◽  
D.A. Calegar ◽  
F.A. Carvalho-Costa ◽  
L.H. Jaeger ◽  
Keyword(s):  
Evolutionary Genetics ◽  
Large Collection ◽  
Chain Reaction ◽  
Rural Regions ◽  
Dna Recovery ◽  
Polymerase Chain ◽  
N 93 ◽  
Initial Processing ◽  
Specific Species

AbstractDespite the reduction in the prevalence of soil-transmitted helminthiases in many regions of the world, morbidity rates remain high in some rural regions. The Kato–Katz technique is a simple, inexpensive and field-applicable tool commonly used for the diagnosis and worm-burden characterization of these infections. Molecular studies have revolutionized our understanding of the epidemiology and evolutionary genetics of parasites. In this study we recovered helminthic DNA from Kato–Katz slides (n = 93) prepared in 2011 in the Brazilian Amazon. We achieved DNA recovery by polymerase chain reaction (PCR) in 84% of cases for Ascaris sp. and 75% of cases for hookworms. The sequencing confirmed the specific species of the amplicons. The slides stored for a few years could be analysed using this methodology, allowing access to DNA from a large collection of samples. We must consider the Kato–Katz thick smears as a source of helminth DNA. This can significantly reduce logistical difficulties in the field in terms of obtaining, preserving, transporting and initial processing of samples.

A Polymerase Chain Reaction (PCR) Method for Sex and Species Determination with Novel Controls for Deoxyribonucleic Acid (DNA) Template Length

1992 ◽  
Vol 37 (1) ◽  
pp. 13207J ◽  
Author(s):  
R. E. Gaensslen ◽  
Karen M. Berka ◽  
Dina A. Grosso ◽  
Gualberto Ruano ◽  
Elaine M. Pagliaro ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Deoxyribonucleic Acid ◽  
Chain Reaction ◽  
Species Determination ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Dna Template

scholarly journals Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7121
Author(s):  
Ankur Naqib ◽  
Silvana Poggi ◽  
Stefan J. Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Microbial Community ◽  
Annealing Temperature ◽  
Degenerate Primer ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain ◽  
Dna Template ◽  
Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

scholarly journals Detection of Leptospira spp. using polymerase chain reaction technique from kidney of Rattus norvegicus from Grenada, West Indies

2019 ◽  
pp. 81-85
Author(s):  
Bhumika Sharma ◽  
Katelyn Thille ◽  
Nia Rametta ◽  
Ravindra Sharma
Keyword(s):  
Polymerase Chain Reaction ◽  
Rattus Norvegicus ◽  
West Indies ◽  
Gene Segment ◽  
Local Alignment ◽  
Active Infection ◽  
Chain Reaction ◽  
Significant Difference ◽  
Leptospira Spp ◽  
Polymerase Chain

Aim: This study aimed to find out the prevalence of active infection of Leptospira spp. in Rattus norvegicus from Grenada, West Indies, through polymerase chain reaction (PCR). Materials and Methods: One hundred and forty-nine rats were trapped, anesthetized and their kidneys collected aseptically. DNA was extracted from the kidney tissue of each rat. PCR was performed targeting LipL32 gene. Eighteen PCR-positive amplicons for LipL32 gene segment were purified and sent for direct sequencing to the sequencing facility of MCLAB (South San Francisco, USA). Results of sequencing were read and interpreted. The prevalence of Leptospira spp. in relation to sex and age was also recorded. Results: All amplified sequences were compared to the sequences present in GenBank using basic local alignment search tool (BLAST) from the online website National Center for Biotechnology Information, the results revealed that six samples had similarity to Leptospira interrogans strain 1399/2016 and eight samples had similarity with Leptospira borgpetersenii serovar Hardjo-bovis strain L49. Of 149 kidney samples, only 14 were positive for Leptospira spp. by PCR giving an incidence of 9.3%. There was no significant difference found in relation to sex and age. Conclusion: This is the first report confirming active infection of Leptospira spp. in Rattus norvegicus in Grenada using PCR. The presence of active infection in rats can be considered as high risk for humans. Further research to understand the epidemiology of leptospirosis in Grenada is suggested.

scholarly journals Polymerase Chain Reaction Multiplication for the Detection of Bacterial Isolates Causing Neonatal Sepsis

2020 ◽  
Vol 8 (A) ◽  
pp. 623-628
Author(s):  
Utari Hartati Suryani ◽  
Andri Rezano ◽  
Yunia Sribudiani
Keyword(s):  
Polymerase Chain Reaction ◽  
Neonatal Sepsis ◽  
Pcr Analysis ◽  
Local Alignment ◽  
Bacterial Isolates ◽  
Chain Reaction ◽  
E Coli ◽  
Initial Design ◽  
Pcr Multiplex ◽  
Polymerase Chain

BACKGROUND: Neonatal sepsis is a clinical syndrome caused by the presence of bacteria in the blood accompanied by symptoms and systemic signs of infection that occurs in the first 4 weeks of life after birth. The process of identifying pathogenic microorganisms is essential in determining the clinical condition in neonatal sepsis. AIM: The study was aimed to develop a multiplex polymerase chain reaction (PCR) method to identify bacterial isolates that cause neonatal sepsis in Indonesia with the main target of optimization of an initial design and PCR optimization. METHODS: This research is an explorative in vitro study for the optimization of an initial design and PCR methods for the detection of the main bacteria that cause sepsis neonatorum in Indonesia, namely, bacteria Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa. The study was conducted at the Biomolecular Laboratory of the Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia. RESULTS: Sequencing carried out and continued with Basic Local Alignment Search Tool (BLAST) sequencing results, it appears that the PCR product that has been produced conforms with the optimization targets that were previously set when doing the primer design. The level of homology found in all four species based on the results of BLAST in a sequence is as follows: K. pneumoniae 94%, P. aeruginosa 99%, E. cloacae 100%, and E. coli 100%. CONCLUSION: PCR multiplex method using design primers and conventional PCR analysis methods (using agarose gel) can be used to detect four species of bacteria that cause neonatal sepsis, namely, K. pneumoniae, P. aeruginosa, E. cloacae, and E. coli.

Polymerase chain reaction identification of a female-specific genetic marker in Arceuthobium americanum (lodgepole pine dwarf mistletoe) and its implications for Arceuthobium sex determination

Botany ◽  
2011 ◽  
Vol 89 (6) ◽  
pp. 369-377 ◽  
Author(s):  
Arvin Dwarka ◽  
Cynthia M. Ross Friedman ◽  
Mairi E. MacKay ◽  
Don Nelson
Keyword(s):  
Polymerase Chain Reaction ◽  
Sex Determination ◽  
Genetic Marker ◽  
Sex Chromosomes ◽  
Local Alignment ◽  
Chain Reaction ◽  
Male And Female ◽  
Arceuthobium Americanum ◽  
Polymerase Chain ◽  
Female Specific

In North America, the most widespread and speciose mistletoe is Arceuthobium M. Bieb. (dwarf mistletoes, Viscaceae), which is a dioecious parasite of conifers. Little is known about its sex determination system, and sex chromosomes have not been identified. A genetic marker for early gender discrimination in Arceuthobium would be useful in the study of sex ratios and sex determination. Here, random amplified polymorphic DNA analysis via the polymerase chain reaction (PCR) was used to investigate genetic differences between genders in Arceuthobium americanum Nutt. ex Engelm. collected near Kamloops, British Columbia and Bélair, Manitoba. A total of 196 10-mer primers were selected for analysis of DNA from isolated male and female A. americanum somatic tissue. A ∼900 bp female-specific DNA fragment was generated with primer OPB-18 (5′-CCACAGCAGT-3′). The fragment was cloned and sequenced. Using GenBank and the basic local alignment search tool alignment software, it was determined that the first ∼300 bp of this DNA sequence shared a high degree of similarity to transposable elements (76%) and a Y-chromosome (male) fragment (75%) in Silene latifolia Poir. Sequence-characterized amplified region primers were then designed. This study has generated an efficient molecular tool to differentiate male and female A. americanum while also providing evidence indicating that A. americanum may have homomorphic, possibly protoheteromorphic, sex chromosomes.

Genetic heterogeneity amongVibrio alginolyticusstrains, and design of a PCR-based identification method usinggyrBgene sequence

2018 ◽  
Vol 64 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Supansa Bunpa ◽  
Mitsuaki Nishibuchi ◽  
Jumroensri Thawonsuwan ◽  
Natthawan Sermwittayawong
Keyword(s):  
Genetic Diversity ◽  
Chain Reaction ◽  
Marine Animals ◽  
Gyrb Sequence ◽  
Highly Sensitive ◽  
Southern Thailand ◽  
Polymerase Chain ◽  
Species Specific ◽  
Dna Template ◽  
Vibrio Spp

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

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