| Pine wood nematode disease is one of the four major tree diseases in the world today.The pathogen is Bursaphelenchus xylophilus,known as pine wood nematode.Due to the devastating damage caused by pine wood nematode disease,the accurate identification of pine wood nematode is considered as the important step in the prevention and control of pine wood nematode disease.At present,there are morphological method,biochemical methods,and traditional molecular techniques for the detection of pine wood nematode.These methods are time-consuming,complicated to operate,and require special instruments.Isothermal amplification technique is a rapid and simple nucleic acid detection method.In this paper,a variety of isothermal amplification techniques were applied to the rapid detection of pine wood nematode,aiming to establish a rapid and accurate method for the detection of pine wood nematode.In the first chapter,the helicase used in sothermal amplification was expressed and purified.Through the literature,the DNA sequence of Tte-UvrD was obtained.The DNA sequence was artificially synthesized,then ligated into the pET-15b plasmid.The Tte-UvrD was expressed by the Escherichia coli BL21 strain.After the enzyme was purified,its activity was tested.The second chapter studies the isothermal amplification technology（HDA）,the loop-mediated isothermal amplification（LAMP）,the improved cross-primer isothermal amplification（CPA）and the denaturation bubble-mediated strand exchange amplification（SEA）assays for the detection of pine wood nematode.After the comparison of the four methods,the SEA was chosen to detect pine wood nematode because of its sensitivity and high-speed.The experiment first used helicase-dependent amplification technology invented by NewEnglandBiolabs to detect pine wood nematode,and 10-13 mol/L pine wood nematode DNA fragment and 4 pg genomic DNA can be detected,but HDA is not sensitive enough to detect single pine wood nematode.In order to improve detection capabilities,isothermal amplification methods that can detect RNA are adopted.According to the literature,Bst DNA polymerase has reverse transcription activity,and the shorter the target sequence is,the higher the reverse transcription activity is.The improved CPA has the ability to detect RNA because of its short target sequence.SEA also has the ability to detect RNA,the results show that the SEA reaction can detect pine wood nematode more effectively.Finally,the SEA reaction was used to detect the pine wood nematode.In third chapter,the SEA rapid detection technology for pine wood nematode can detect10-13 mol/L pine wood nematode DNA fragment in 40 min.,and 400 fg genomic DNA could be detected.The nucleic acids of pine wood nematode sample was extracted at 95°C for 5min and in this way SEA can detect single nematode.The established SEA rapid detection technology for pine wood nematode can detect single nematode in 50 min,which meets the rapid detection requirements of pine wood nematode,so the technology can be widely used in the inspection and quarantine of inbound and outbound timber,early diagnosis and suspected sample identification of pine wood nematode disease. |
