Essay On Soil Detection In Soil

853 Words4 Pages
PMTV detection in soil

Soil Samples collection and preparation

Soil samples used in this study were collected from a PMTV infested potato field in North Dakota in 2016 and 2017. Additionally, comparable soil samples were collected from the nearby field in 2017 where PMTV has never been detected. Collected soil was dried at ambient temperature then pulverized and stored at cold room (4 ℃). Subsamples of soils were sterilized by autoclaving twice at 121 C for 60 minutes before storing at 4 ℃.

Soil was artificially contaminated with PMTV using Sporosori of Spongospora Subterranea (Vector of PMTV). Sporosori of S. Subterranea used in this study was harvested from the surface of infected potato tuber by scraping with a scalpel followed by
…show more content…
2004). One- tube single step RT-PCR assay was carried out in 50 µL reaction mixture using QIAGEN OneStep RT- PCR kit (Qiagen,Ca). RT-PCR was carried out using a thermal cycler (T100 thermal cycler, Bio-Rad) to amplify cDNA fragments using the following conditions: RT at 50 °C for 30 min for reverse transcription followed by initial PCR activation for 15 min at 95 °C; 40 cycles of PCR, with each cycle at 94 °C for 60 s, 52 °C (while using PMTV2F and PMTV3 R)or 57°C ( while using C819 and H360 ) for 60 s, and 72 °C for 60 s; and a final extension at 70°C for 10 min. RT-PCR assay products were resolved by standard electrophoresis technique in 1% agarose gels pre- stained with gel red and the DNA bands were visualized under a UV-transilluminator. PCR products were cleaned by QIAquick PCR purification Kit (Qiagen) and subjected to Sanger sequencing (Retrogen, Inc. San Diego, and CA). The sensitivity of RT-PCR technique for detection of PMTV from soil extracted RNA was determined by using total RNA extracted from series of spiked soils.

Real time reverse transcription polymerase chain reaction (qRT-PCR)
Attempt was also made to detect PMTV from the soil extracted RNA using qRT-PCR. The primers and probes were designed targeting coat protein read through protein gene with the help of sequences obtained from
Get Access