scholarly journals EFEKTIVITAS TRANSFER DAN ANALISIS EKSPRESI GEN IMUNOGENIK TAHAN KOI HERPES VIRUS (KHV) PADA IKAN MAS (Cyprinus carpio)

2014 ◽  
Vol 9 (1) ◽  
pp. 15
Author(s):  
Khairul Syahputra ◽  
Didik Ariyanto ◽  
Erma Primanita Hayuningtyas ◽  
Lamanto Lamanto
Keyword(s):  
Reverse Transcriptase ◽  
Cyprinus Carpio ◽  
Herpes Virus ◽  
Rt Pcr ◽  
Square Wave ◽  
Reverse Transcriptase Pcr ◽  
Herpès Virus

Penelitian transfer gen imunogenik tahan KHV (krt-GP11) pada ikan mas telah dilakukan dengan metode elektroporasi sperma menggunakan konsentrasi DNA yang berbeda. Penelitian ini bertujuan untuk mengetahui konsentrasi DNA optimal yang efektif digunakan dalam transfer gen pada ikan mas. Sperma dielektroporasi menggunakan tipe kejutan square wave dengan voltase 50 V dan jumlah kejutan tiga kali. Konsentrasi DNA yang digunakan adalah 10 μg/mL, 50 μg/mL, dan 100 μg/mL. Deteksi transgen pada sperma, embrio, dan larva dilakukan dengan metode PCR menggunakan primer spesifik untuk gen krt-GP11. Ekspresi transgen pada embrio dan larva dianalisis secara semi-kuantitatif dengan metode reverse transcriptase PCR (RT-PCR). Hasil penelitian menunjukkan bahwa gen krt-GP11 terdeteksi pada sperma, embrio, dan larva. Pemberian konsentrasi DNA 10 μg/mL lebih efektif digunakan dalam transfer gen krt-GP11 pada ikan mas, sedangkan peningkatan konsentrasi DNA yang digunakan tidak memberikan hasil yang berbeda terhadap keberhasilan transfer gen pada ikan mas. Ekspresi gen krt-GP11 yang berhasil diintroduksikan pada ikan mas baru dapat teramati dengan baik pada fase embrio.

scholarly journals Artemia sp. as a DNA vaccine vector for common carp Cyprinus carpio larvae

2014 ◽  
Vol 12 (1) ◽  
pp. 54
Author(s):  
Sri Nuryati ◽  
Sekar Sulistyaning Hadiwibowo ◽  
. Alimuddin
Keyword(s):  
Dna Vaccine ◽  
Common Carp ◽  
Cyprinus Carpio ◽  
Herpes Virus ◽  
Oral Vaccine ◽  
Viral Disease ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Glycoprotein Gene ◽  
Herpès Virus

<p class="NoParagraphStyle" align="center"><strong>ABSTRACT</strong></p><p class="NoParagraphStyle" align="center"> </p><p class="NoParagraphStyle">Koi herpes virus (KHV) is one of the most common impetuses for disease on common carp <em>Cyprinus carpio</em>. Generally, viral disease is difficult to cure because virus is intra-cellular parasite, that virus survives, multiplies, and lives only if it on the host cell. Oral vaccine delivery through <em>Artemia</em> sp. is of one alternative way to overcome this problem. This experiment was carried out by analysis DNA vaccine expression encoding of glycoprotein gene (GP-11) on <em>C. carpio</em>. Bacteria containing plasmid Krt-GP-11 as vaccine is served through <em>Artemia </em>sp. as a vector. <em>Artemia</em> sp. was given for one and two times a week to three weeks old common carp. Organs of fish fed by <em>Artemia</em> sp. were analyzed every three days after vaccination. The expression of GP-11 in kidney in each treatment is also observed by the use of RT-PCR method, within ten days after vaccination. The experiment showed that dose of DNA vaccine in whole bacteria could be expressed is 10<sup>6</sup> cfu/mL in a once or twice provisions a week. DNA vaccine could be detected in three organs. RT-PCR analysis also showed that the expression of GP-11 can be detected in all tested organs. In conclusion, <em>Artemia</em> sp. can be used as a vector to carry plasmid GP-11 vaccine for common carp <em>Cyprinus carpio</em> larvae.</p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle">Keywords: DNA vaccine, KHV, <em>Artemia</em> sp., common carp</p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle" align="center"><strong>ABSTRAK</strong></p><p class="NoParagraphStyle"> </p><p class="NoParagraphStyle">Salah satu penyakit pada ikan mas (<em>Cyprinus carpio</em>) yang disebabkan oleh virus adalah <em>koi herpes virus</em> (KHV). Penyakit yang disebabkan oleh virus umumnya sulit untuk disembuhkan karena virus merupakan parasit intraseluler, yaitu virus hanya dapat hidup, bertahan hidup, dan memperbanyak diri di dalam sel inang. Metode pemberian vaksin DNA secara oral melalui <em>Artemia</em> sp. merupakan salah satu alternatif pengobatan yang diharapkan dapat menangani permasalahan penyakit pada ikan yang disebabkan oleh virus. Pada penelitian ini dilakukan uji ekspresi vaksin DNA yang menyandikan glikoprotein 11 (GP-11) pada ikan mas. Bakteri yang mengandung plasmid Krt-GP-11 sebagai vaksin diberikan melalui <em>Artemia</em> sp. sebagai pembawa vaksin. Pemberian <em>Artemia</em> sp. dilakukan satu dan dua kali seminggu pada ikan mas umur tiga minggu. Keberadaan DNA vaksin di usus, ginjal, dan insang dianalisis menggunakan metode PCR. Organ diambil setiap tiga hari setelah pemberian vaksin. Ekspresi gen GP-11 juga diamati pada organ ginjal di setiap perlakuan dengan menggunakan metode RT-PCR, pada sepuluh hari setelah pemberian vaksin. Hasil penelitian menunjukkan bahwa DNA vaksin yang diberikan dengan dosis 10<sup>6</sup> cfu/mL pada perlakuan satu dan dua kali seminggu dapat terdeteksi pada ketiga organ. Hasil RT-PCR menunjukkan bahwa ekspresi GP-11 dapat terdeteksi pada semua organ uji di setiap perlakuan. Dengan demikian <em>Artemia</em> sp. dapat digunakan sebagai vektor pembawa vaksin plasmid GP-11 dengan frekuensi pemberian vaksin untuk larva ikan mas.</p><p class="NoParagraphStyle"> </p><p>Kata kunci: vaksin DNA, KHV, <em>Artemia</em> sp., ikan mas</p>

scholarly journals First Genome Sequence of Newcastle Disease Virus of Genotype VIIi from Jordan

2018 ◽  
Vol 7 (23) ◽  
Author(s):  
Mustafa Ababneh ◽  
Helena L. Ferreira ◽  
Mohammad Khalifeh ◽  
David L. Suarez ◽  
Claudio L. Afonso
Keyword(s):  
Reverse Transcriptase ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Genome Sequence ◽  
Illumina Sequencing ◽  
Newcastle Disease ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Chicken Spleen

Newcastle disease virus (NDV) was detected by reverse transcriptase PCR (RT-PCR) from total RNA isolated from a chicken spleen of a backyard flock in Jordan. The complete coding genome sequence of NDV/chicken/Jordan/J11-spleen/2018 was obtained with MiSeq (Illumina) sequencing.

A Reverse Transcriptase PCR Technique for the Detection and Viability Assessment of Kluyveromyces marxianus in Yoghurt

2006 ◽  
Vol 69 (9) ◽  
pp. 2210-2216 ◽  
Author(s):  
MARÍA BELÉN MAYORAL ◽  
ROSARIO MARTÍN ◽  
PABLO E. HERNÁNDEZ ◽  
ISABEL GONZÁLEZ ◽  
TERESA GARCÍA
Keyword(s):  
Reverse Transcriptase ◽  
Kluyveromyces Marxianus ◽  
18S Rrna ◽  
Yeast Cells ◽  
Rt Pcr ◽  
Viability Assessment ◽  
Reverse Transcriptase Pcr ◽  
Low Concentrations ◽  
Pcr Method ◽  
High Level

A fast and sensitive reverse transcriptase PCR (RT-PCR) method was developed for the detection of viable Kluyveromyces marxianus in yoghurt. Yeast-specific primers were used with the RT-PCR to evaluate the suitability of 18S rRNA as a target for the detection of viable yeasts in pure culture and yoghurt. The RT-PCR assay was able to detect down to 102 CFU ml−1 in yoghurt samples contaminated with viable yeast cells. Application of the RT-PCR method to commercial yoghurt samples demonstrated the utility of this technique for detection of low concentrations of viable yeast cells in naturally contaminated dairy products. The 18S rRNA molecule is an appropriate target for cell viability assessment because of its limited persistence after cell death and the resultant high level of sensitivity of the assay.

scholarly journals Detection of Mycobacterium tuberculosis Group Organisms in Human and Mouse Joint Tissue by Reverse Transcriptase PCR: Prevalence in Diseased Synovial Tissue Suggests Lack of Specific Association with Rheumatoid Arthritis

2001 ◽  
Vol 69 (3) ◽  
pp. 1821-1831 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Andrew A. McColm ◽  
Julie A. Bagshaw ◽  
Richard Reece ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mycobacterium Tuberculosis ◽  
Synovial Fluid ◽  
Reverse Transcriptase ◽  
Synovial Tissue ◽  
Rt Pcr ◽  
Diverse Range ◽  
Susceptible Host ◽  
Joint Tissue ◽  
Reverse Transcriptase Pcr

ABSTRACT Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model ofM. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosisinfection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.

scholarly journals Two variants of quantitative reverse transcriptase PCR used to show differential expression of α-, β- and γ-fibrinogen genes in rat liver lobes

1997 ◽  
Vol 321 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Junlong ZHANG ◽  
Mina DESAI ◽  
Susan E. OZANNE ◽  
Cora DOHERTY ◽  
C. Nicholas HALES ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rat Liver ◽  
Coefficient Of Variation ◽  
Copy Number ◽  
Internal Standard ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Mrna Copy Number ◽  
Sensitive Method

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of α-, β- and γ-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (α-fibrinogen F = 14.64, P = 0.0003; β-fibrinogen F = 3.74, P = 0.04; γ-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

scholarly journals Use of Reverse Transcriptase PCR in Early Diagnosis of Rift Valley Fever

2002 ◽  
Vol 9 (3) ◽  
pp. 713-715 ◽  
Author(s):  
A. A. Sall ◽  
E. A. Macondo ◽  
O. K. Sène ◽  
M. Diagne ◽  
R. Sylla ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Virus Isolation ◽  
Immunoglobulin M ◽  
Antibody Detection ◽  
Rt Pcr ◽  
Valley Fever ◽  
Reverse Transcriptase Pcr ◽  
Sensitive Tool

ABSTRACT Reverse transcriptase PCR (RT-PCR) for diagnosis of Rift Valley fever (RVF) was evaluated by using 293 human and animal sera sampled during an RVF outbreak in Mauritania in 1998. Results of the RT-PCR diagnostic method were compared with those of virus isolation (VI) and detection of immunoglobulin M (IgM) antibodies. Our results showed that RT-PCR is a specific, sensitive tool for RVF diagnosis in the early phase of the disease and that its results do not differ significantly from those obtained by VI. Moreover, the combined results of RT-PCR and IgM antibody detection were in 100% concordance with the results of VI.

scholarly journals Diagnóstico etiológico de enfermidades do sistema nervoso central de equinos no Estado de Minas Gerais, Brasil

2015 ◽  
Vol 67 (2) ◽  
pp. 391-399 ◽  
Author(s):  
E.A. Costa ◽  
R. Rosa ◽  
T.S. Oliveira ◽  
R. Furtini ◽  
A.A. Fonseca Júnior ◽  
...  
Keyword(s):  
Herpes Virus ◽  
Minas Gerais ◽  
Rt Pcr ◽  
Saint Louis ◽  
Herpès Virus

O Brasil possui o quarto maior rebanho equino do mundo, e o Estado de Minas Gerais detém o maior número de equinos do país. Portanto, um diagnóstico preciso das doenças neurológicas dos equinos é prioridade no estado. Sendo assim, o objetivo deste estudo foi identificar, utilizando a Reação em Cadeia pela Polimerase (PCR), os agentes infecciosos responsáveis por enfermidades que afetam o sistema nervoso central (SNC) de equinos. De janeiro de 2009 a janeiro de 2011, foi realizado um levantamento dos casos de encefalites e encefalomielites em equinos no Estado de Minas Gerais, utilizando-se amostras de SNC de equinos que morreram com sinais neurológicos. Das 217 amostras de SNC, 47 (21,7%) foram positivas para o vírus da raiva pelo método de imunofluorescência indireta e inoculação em camundongos. Nas 170 amostras negativas para o vírus da raiva, o herpes-vírus equino-1 (EHV-1) foi diagnosticado em 20 (11,8%) e o herpes-vírus suíno-1 (SHV-1), em uma amostra por meio de PCR, e o vírus encefalite de Saint Louis (SLEV), em outra amostra, através de transcrição reversa (RT) e PCR (RT-PCR). Constatou-se que o vírus da raiva é o principal agente causador de encefalite em equinos, apesar do crescente número de casos de encefalomielite associados ao EHV-1 no Estado de Minas Gerais.

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