scholarly journals Direct in situ reverse transcriptase-polymerase chain reaction

2001 ◽  
Vol 281 (2) ◽  
pp. C726-C732 ◽  
Author(s):  
Rajesh Kher ◽  
Robert Bacallao
Keyword(s):  
Reverse Transcriptase ◽  
Restriction Enzymes ◽  
Cellular Level ◽  
Detection Sensitivity ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
A Cell ◽  
Polymerase Chain ◽  
In Situ Detection

In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.

scholarly journals In Situ Detection of Benzimidazole Resistance in Field Isolates of Venturia inaequalis in Indiana

Plant Disease ◽  
2010 ◽  
Vol 94 (6) ◽  
pp. 744-750 ◽  
Author(s):  
Kacie L. Quello ◽  
Kimberly S. Chapman ◽  
Janna L. Beckerman
Keyword(s):  
Venturia Inaequalis ◽  
Apple Scab ◽  
Restriction Enzymes ◽  
Fungicide Sensitivity ◽  
Benzimidazole Resistance ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
In Situ Detection ◽  
Moderately Resistant

Venturia inaequalis, the causal agent of apple scab, infects both commercial apples and ornamental crabapples. We found four classes of benzimidazole fungicide sensitivity in the Indiana population: sensitive (S) isolates unable to grow on 0.5 μg active ingredient (a.i.)/ml; low resistant (LR) isolates that grew at 0.5 μg a.i./ml, but not at 5 μg a.i./ml; moderately resistant (MR) isolates that grew at 5 μg a.i./ml, but not at 50 μg a.i./ml; and very highly resistant (VHR) isolates that grew rapidly at 50 μg a.i./ml. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the β-tubulin gene with two restriction enzymes, BstUI and Cac8I, enabled us to rapidly identify benzimidazole resistance among all tested isolates. Sixty-nine percent of the resistant isolates tested possessed the BstUI RFLP at codon 198 that corresponds to VHR, and the remaining LR and MR isolates possessed the Cac8I RFLP corresponding to a newly identified resistance allele at codon L240F. Combined, PCR-RFLP correctly identified the resistance status of all isolates tested to date. The preponderance of benzimidazole-resistant isolates from commercial apple orchards and their absence in the landscape on ornamental crabapple suggests that two distinct populations of V. inaequalis coexist in Indiana.

scholarly journals MONITORING PENYAKIT WSSV PADA BUDIDAYA UDANG WINDU (Penaeus monodon) DI TAMBAK TRADISIONAL KOTA TARAKAN

2020 ◽  
Vol 12 (2) ◽  
pp. 89-95
Author(s):  
Rukisah Rukisah ◽  
Gloria Ika Satriani ◽  
Rosmawati Rasyid
Keyword(s):  
White Spot Syndrome Virus ◽  
Penaeus Monodon ◽  
White Spot ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Polymerase Chain ◽  
White Spot Syndrome

ABSTRAKPenyakit WSSV (white spot syndrome virus) pertama kali dilaporkan menyebabkan wabah mematikan pada udang Penaeus japonicas di Jepang pada tahun 1993 (Nakano et al., 1994) kemudian menyebar hampir ke seluruh wilayah Asia, termasuk Indonesia. Hingga saat ini, WSSV masih menjadi penyebab utama kegagalan panen pada tambak tradisional Kota Tarakan. Virus ini merupakan famili Nimaviridae dari genus Whispovirus yang menyerang udang pada semua stadia dan merupakan patogen mematikan bagi semua jenis udang penaeid. Tujuan penelitian ini untuk mengetahui kondisi WSSV di kawasan tambak-tambak tradisional Tarakan (wilayah barat, timur, dan utara) sehingga dapat dilakukan tindakan penanggulangan terhadap penularan WSSV di Kota Tarakan. Antisipasi terhadap penyebaran WSSV dilakukan secara vertikal maupun horizontal. Secara vertikal melalui genetik, sedangkan horizontal melalui rantai makanan, faktor transmisi, dan reservoir infeksi. Penelitian dilakukan di semester awal (Januari-Juni) tahun 2016 menggunakan metode sampling populasi secara acak dan selektif. Sample udang windu (Penaeus monodon) dianalisis kualitatif secara deskriptif menggunakan teknik PCR (polymerase chain reactions) berdasarkan OIE Protocol (Office International Des Epizooties) di Stasiun Karantina Kelas II Tarakan, dan parameter penunjang berupa data kualitas air diukur secara in situ. Hasil penelitian bahwa seluruh sampel yang dianalisis pada 146 F1/R1 Primers First Step menunjukan pita DNA kurang dari 1447 bp hasilnya negatif WSSV, namun pada tahap kedua 146 F2/R2 Primers Nested terdapat satu sampel menghasilkan pita DNA 941 bp hasilnya positif WSSV. Data kualitas air masih sesuai persyaratan baku mutu budidaya windu (SNI 7310-2009). Pada akhir penelitian, tidak ditemukan satu pun tambak tradisional di lokasi penelitian yang mengalami kegagalan panen.Kata kunci: WSSV, Penaeus monodon, PCR, Tambak Tradisional

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