scholarly journals Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera)

2011 ◽  
Vol 4 (1) ◽  
Author(s):  
Yolanda Bel ◽  
Juan Ferré ◽  
Baltasar Escriche
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Duplication ◽  
Real Time ◽  
Real Time Pcr ◽  
Ostrinia Nubilalis ◽  
Gene Dosage ◽  
Sybr Green ◽  
Quantitative Real Time Pcr ◽  
Sybr Green Detection

The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

2006 ◽  
Vol 65 (3) ◽  
pp. 476-487 ◽  
Author(s):  
Miguel A. Providenti ◽  
Jason M. O'Brien ◽  
Robyn J. Ewing ◽  
E. Suzanne Paterson ◽  
Myron L. Smith
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Sybr Green ◽  
Quantitative Real Time Pcr ◽  
Genetic Elements

scholarly journals Gene Dosage by Quantitative Real-Time PCR

BioTechniques ◽  
1999 ◽  
Vol 27 (2) ◽  
pp. 228-232 ◽  
Author(s):  
J.-L. Boulay ◽  
J. Reuter ◽  
R. Ritschard ◽  
L. Terracciano ◽  
R. Herrmann ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gene Dosage ◽  
Quantitative Real Time Pcr

Investigation of the Interference of Carbon Nanomaterials with SYBR Green I-Based Real-Time PCR

2013 ◽  
Vol 785-786 ◽  
pp. 550-555
Author(s):  
Fu Ming Sang ◽  
Yuan Sun ◽  
Zhong Xu ◽  
Yu Shi Wang ◽  
Zhi Zhou Zhang
Keyword(s):  
Fluorescence Quenching ◽  
Real Time ◽  
Melting Temperature ◽  
Real Time Pcr ◽  
Carbon Nanomaterials ◽  
Pcr Amplification ◽  
Sybr Green ◽  
Sybr Green I ◽  
Amplification Efficiency ◽  
Quantitative Real Time Pcr

Some carbon nanomaterials have been proved to be able to improve the PCR amplification efficiency. If used in quantitative real-time PCR (qPCR), these nanomaterials must be tested whether fluorescence processing is interfered after they are added in the PCR system. In this study, 76 different carbon nanomaterials were tested in SYBR Green I-based qPCR, and the results demonstrated that about half carbon nanomaterials tested in this study could alter the PCR amplification profile probably due to the fluorescence quenching. Surprisingly, lower concentrations of nanomaterials led to more slight interference with the melting temperature.

scholarly journals Rapid method for identification of transgenic fish zygosity

2007 ◽  
Vol 6 (2) ◽  
pp. 177
Author(s):  
. Alimuddin ◽  
G. Yoshizaki ◽  
O. Carman
Keyword(s):  
Real Time ◽  
Danio Rerio ◽  
Real Time Pcr ◽  
Detection System ◽  
Transgenic Fish ◽  
Sybr Green ◽  
Wild Type ◽  
Quantitative Real Time Pcr ◽  
Oncorhynchus Masou ◽  
Cycle Threshold

<p>Identification of zygosity in transgenik fish is normally achieved by PCR analysis with genomic DNA template extracted from the tissue of progenies which are derived by mating the transgenic fish and wild-type counterpart.  This method needs relatively large amounts of fish material and is time- and labor-intensive. New approaches addressing this problem could be of great help for fish biotechnologists.  In this experiment, we applied a quantitative real-time PCR (qr-PCR) method to analyze zygosity in a stable line of transgenic zebrafish (<em>Danio rerio</em>) carrying masu salmon, <em>Oncorhynchus masou</em> D6-desaturase-like gene. The qr-PCR was performed using iQ SYBR Green Supermix in the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, USA).  Data were analyzed using the comparative cycle threshold method.  The results demonstrated a clear-cut identification of all transgenic fish (<em>n</em>=20) classified as a homozygous or heterozygous.  Mating of those fish with wild-type had revealed transgene transmission to the offspring following expected Mendelian laws. Thus, we found that the qTR-PCR to be effective for a rapid and precise determination of zygosity in transgenic fish. This technique could be useful in the establishment of breeding programs for mass transgenic fish production and in experiments in which zygosity effect could have a functional impact.</p> <p>Keywords: quantitative real-time PCR; zygosity; transgenic fish; mass production</p> <p> </p> <p>ABSTRAK</p> <p>Identifikasi sigositas ikan transgenik biasanya dilakukan menggunakan analisa PCR dengan cetakan DNA genomik yang diekstraksi dari jaringan ikan hasil persilangan antara ikan transgenik dan ikan normal.   Metode ini memerlukan ikan dalam jumlah yang banyak, dan juga waktu serta tenaga.  Pendekatan baru untuk mengatasi masalah tersebut akan memberikan manfaat besar kepada peneliti bioteknologi perikanan.  Pada penelitian ini, kami menggunakan metode PCR real-time kuantitatif (krt-PCR) untuk menganalisa sigositas pada satu strain ikan zebra (<em>Danio rerio</em>)<em> </em>transgenik yang membawa gen D6-desaturase-like dari ikan salmon masu, <em>Oncorhynchus masou</em>.<em>  </em>krt-PCR dilakukan menggunakan <em>iQ SYBR Green Supermix</em> pada mesin <em>iCycler iQ Real-time PCR Detection system</em> (Bio-Rad Laboratories, USA).  Data dianalisis menggunakan metode pembandingan nilai <em>cycle threshold</em>.  Hasil penelitian menunjukkan bahwa semua ikan transgenik (<em>n</em>=20) yang diidentifikasi dapat diklasifikasikan secara jelas sebagai ikan homosigot atau heterosigot.  Persilangan antara ikan transgenik tersebut dengan ikan normal menunjukkan transmisi transgen ke keturunannya mengikuti hukum segregasi Mendel.  Dengan demikian, metode krt-PCR adalah efektif untuk penentuan sigositas secara cepat dan tepat pada ikan transgenik.  Teknik ini dapat berguna dalam program produksi ikan transgenik secara massal dan dalam percobaan dimana faktor sigositas memberikan pengaruh nyata.</p> Kata kunci: kuantitatif real-time PCR; sigositas, ikan transgenik; produksi massal

scholarly journals SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

2020 ◽  
Author(s):  
Wallop Jakkul ◽  
Kittipong Chaisiri ◽  
Naowarat Saralamba ◽  
Yanin Limpanont ◽  
Sirilak Dusitsittipon ◽  
...  
Keyword(s):  
Intermediate Host ◽  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Eosinophilic Meningitis ◽  
Quantitative Real Time Pcr ◽  
The Third ◽  
Snail Intermediate Host ◽  
Third Stage ◽  
Qpcr Method

Abstract Background: Angiostrongylus cantonensis is a well-known pathogen causing human angiostrongyliasis eosinophilic meningitis. Humans, as accidental hosts, are infected by eating undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. Recently, the two species have been reported to have overlapping distributions in the same endemic area, particularly in the Indochina region. Because of their similar morphological characteristics, misidentification often occurs, particularly of the third-stage larva in the snail intermediate host. Methods: We designed species-specific primers to mitochondrial cytochrome b, which was used as a genetic marker. SYBR-green quantitative real-time PCR (qPCR) was employed to quantitatively detect and identify the third-stage larvae and tissue debris in the cerebrospinal fluid (CSF) of a patient, and to quantify third-stage larvae in the snail Achatina fulica collected from the field.Results: The newly designed primers were highly specific and sensitive, even when using conventional PCR. SYBR green qPCR quantitatively detected around 10−4 ng of genomic DNA from one larva and facilitated the specific detection and identification of parasitic genetic material from the CSF of a patient with angiostrongyliasis. The method also estimated the number of larvae in A. fulica and revealed that the primary source of Angiostrongylus infection in the King Rama IX public park study area was A. malaysiensis; although, the two Angiostrongylus species each infected 10% of the snails. Conclusions: Our SYBR green qPCR method is a useful and inexpensive technique for parasite identification and has sufficient sensitivity and specificity to detect a single larva and simultaneously discriminate between A. cantonensis and A. malaysiensis. The number of larvae infecting or co-infecting the snail intermediate host can also be estimated. In future research, this qPCR method could be employed in a molecular survey of A. cantonensis and A. malaysiensis occurrence within intermediate and definitive hosts. The technique should also be applied in a study analyzing CSF specimens from patients with eosinophilic meningitis to assess the usefulness of the method for clinical diagnosis.

Sign in / Sign up

Export Citation Format

Share Document