Detection of Tomato Yellow Leaf Curl Thailand Virus by PCR Without DNA Extraction

Abstract

We report the simple and rapid method for detection of tomato yellow leaf curl Thailand virus (TYLCTHV) based on the direct capture of virus particles to the surface of a polymerase chain reaction (PCR) tube. This method allowed PCR without the time-consuming procedures of DNA extraction from infected plant tissue. A small amount of tomato tissue (approximately 10 mg) was ground in extraction buffer to release viruses from plant tissues. The constituents of the plant extract that might inhibit PCR activity were discarded by washing the tube with PBST buffer before adding the PCR mixture to the tube. This method was used for detection of TYLCTHV with plant sap solution diluted up to 1:20,000 and was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method. In addition, this method can be used for detection of TYLCTHV in viruliferous whiteflies. The PCR tubes with captured TYLCTHV could be used for PCR, after storage at 4 degrees C for 4 wk. The method presented here was used for detection of begomoviruses in cucurbit and pepper. In addition, this method was effectively used to detect papaya ringspot virus in papaya and zucchini yellow mosaic virus in cucumber by reverse transcriptase (RT)-PCR.

Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction

“…DNA extraction efficiency varied from kit to kit and test to test, and the extraction process not only was time consuming but also tended to increase the risk of carry-over contamination. Direct PCR without DNA extraction for qualitative use has been reported in several studies (Castley et al, 2005;Ieamkhang et al, 2005). In the present study, our aim was to quantify human hepatitis B virus (HBV) DNA by direct real-time PCR from serum without a DNA extraction procedure.…”

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

“…Tomato yellow leaf curl Thailand virus could be detected by a direct PCR method in 1 : 20 000 diluted sap from 10 mg tissue (Leamkhang et al . ). As only 1 mg tissue was used in the present method, its detection limit appears to be about twofold higher than this method.…”

“…Tomato yellow leaf curl Thailand virus was detected by capturing virus particles to the surface of PCR tube without DNA extraction; however, the method required tissue grinding in phosphate buffer (Leamkhang et al . ). Tissue blotting PCR assay (also known as print capture PCR) was used for detecting plum pox virus (PPV) from infected plants without grinding the sample (Olmos et al .…”

Immunocapture Loop Mediated Isothermal Amplification for Rapid Detection of Tomato Yellow Leaf curl Virus (TYLCV) without DNA Extraction

“…As a result, fast, costeffective, reliable and sensitive indexing techniques are requisite tools to determine the virus status, particularly during early stages of viral infection, double antibody sandwich enzyme linked immune sorbent assay (DAS-ELISA), in this context, being considered as one of the first detection approaches [24][25][26]. Some alternative approaches were gradually developed including TAS-ELISA (Triple Antibody Sandwich ELISA) [4,27] and molecular methods including PCR [28], dot blot hybridization [3], tissue blotting immunobinding assay (TBIA) [24], Hybridization with probe [12] and LAMP [29,30], all of which were unfortunately time consuming and require expensive or carcinogenic materials to visualize DNA amplification [31]. Meanwhile, extraction of DNA is another exhausting task, accomplished commonly under various protocols, all of which are typically accompanied by some drawbacks.…”