A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta

Abstract

Significance and Impact of the Study: Identification of begomoviruses by serology is not suitable due to difficulty in preparing high titre and specific antisera. Begomoviruses are routinely detected by PCRbased techniques using universal or specific primers. However, it is a prerequisite to isolate pure DNA from the samples before PCR. DNA extraction from some plants such as jute, mesta is very difficult due to the presence of mucilage and other impurities. Therefore, we have developed a method of direct PCR without DNA extraction for detection of begomoviruses from these crops. It is the first report of a direct PCR method in jute and mesta. AbstractWe have developed a simple method of direct PCR (dPCR) without timeconsuming procedures of DNA extraction by directly using the leaf bits for rapid detection of begomoviruses in jute and mesta. The leaf bits were treated with a lysis buffer for 35 min, and the lysate was used as PCR template. Different components and their concentration in lysis buffer systems were optimized and the optimal buffer system composed of 20 mmol l À1 tris (hydroxymethyl aminomethane (Tris)-Cl (pH 8Á0), 1Á5 mmol l À1ethylenediaminetetraacetic acid (EDTA) (pH 8Á0), 1Á4 mol l À1 NaCl and 200 lg/mL Proteinase K. Further, 3% PVP (w/v) and b-marcaptoethanol (1% v/v) were additionally added into the buffer in case of jute. Under optimized PCR conditions, both viral DNA as well as plant (jute and mesta) genomic DNA were amplified from the lysate. dPCR required fewer reagents and less incubation time reducing both time and cost of detection.

Rapid Real-time Viral Load Estimation Technique for chilli leaf curl virus and its validation in different chilli genotypes from Eastern Himalayan Plains.

“…Many studies in the past have used direct PCR ampli cation protocols from plant tissues [22], [31], [32] either to test the e ciency of different marker system or to detect infectious pathogens. In case of viruses, detection directly from saps of infected leaves is often preferred to eliminate the lengthy and expensive DNA extraction step.…”

Mutagenesis of both prophenoloxidases in the fall armyworm induces major defects in metamorphosis

“…To identify these wanted individuals, the G2 6 th -instar larvae were individually genotyped. Five µL of freshly collected 6 th -instar larvae hemolymph were mixed with 15 µL of lysis buffer adapted from Biswas et al (2014) [20 mmol/L tris (hydroxymethyl aminomethane (Tris)-HCl (pH 8), 1.5 mmol/L ethylenediaminetetraacetic acid (EDTA) (pH 80), 1.4 mol/L NaCl and 200 µg/mL Proteinase K], incubated for 30 min at 65°C and then 2 min at 98°C. One µL of this mixture was used as template for the PCR and one of the above strategies was applied to identify homozygous mutant individuals.…”

Deteksi Brucella abortus dari Sampel Darah-Utuh dengan Uji Polymerase Chain Reaction Tanpa Ekstraksi DNA

“…Saat ini metode amplifikasi tanpa ekstraksi (pemurnian) DNA dari berbagai jenis sampel seperti darah-utuh, tanaman, jaringan hewan atau manusia telah dikembangkan dan berbagai perangkatnya telah tersedia secara komersial (Biswas et al, 2013). Metode ini menawarkan cara yang lebih baik untuk mengurangi waktu penanganan sampel, dana yang diperlukan untuk pemurnian template (cetakan) dan risiko penyebaran mikroorganisme infeksius seperti Brucella spp.…”

“…Dari beberapa hasil penelitian sebelumnya, mekanisme kerja dari senyawa-senyawa aktif yang terkandung di dalam perangkat yang tersedia secara komersial dipercaya mengakibatkan netralisasi zat penghambat yang bertanggung jawab terjadinya pengikatan terhadap enzim DNA polimerase dan/atau cetakan DNA. Terikatnya faktor penghambat oleh senyawa di dalam perangkat akan membebaskan enzim DNA polimerase dan/atau cetakan DNA untuk digunakan dalam proses amplifikasi (Biswas et al, 2013). Pada prinsipnya efektifitas uji PCR secara langsung terletak pada penggunaan sampel DNA yang berkualitas tinggi dan enzim DNA polymerase yang resisten terhadap faktor penghambat yang terkandung di dalam sampel darah merupakan solusinya (Miura et al, 2013;Werblow et al, 2016).…”