Molecular identification method for xiphinama brevicolle

A nematode and detection method technology, applied in the field of quarantine, can solve the problems such as the difficulty of molecular classification and identification, and achieve the effect of low detection limit and good specificity

Inactive Publication Date: 2011-08-17
中华人民共和国珠海出入境检验检疫局
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  • Abstract
  • Description
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Problems solved by technology

[0019] Although there have been great breakthroughs in the molecular classification and identification of plant nematodes, so far all nematode cla

Method used

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  • Molecular identification method for xiphinama brevicolle
  • Molecular identification method for xiphinama brevicolle

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Experimental program
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Embodiment Construction

[0042] Below in conjunction with embodiment, further illustrate the present invention.

[0043] Prepare the extract: the extract contains 2.5 mmol / L of DTT, 1.125% Tween 20, 0.025% gelatin, 2.5 times of PCR buffer, and the balance is water. The PCR buffer contains 125 mmol / L KCl, 25 mmol / L Tris-HCl at pH=8.3, 3.75 mmol / L MgCl 2 , the balance is water;

[0044] Pre-cool the prepared extract to 4°C, and drop it onto the glass slide, and place the nematodes to be tested in the extract;

[0045] Cut the nematode into segments, pipette 8 μL of the segmented nematode suspension into an Eppendorf tube containing 10 μL of sterile water, then add 2 μL of pre-cooled 2mg / mL proteinase K to the tube to make the total volume 20 μL, and quickly Place in -70°C freezer for more than 10 minutes;

[0046] The frozen Eppendorf tube was kept at 65°C for 60 min, 95°C for 10 min, and centrifuged at 12,000 g for 0.5 min to obtain a nematode DNA suspension.

[0047] The total volume of the real-t...

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Abstract

The invention discloses a molecular identification method for xiphinama brevicolle. The method comprises the following steps of: extracting the DNA of xiphinama brevicolle to be tested; performing real-time polymerase chain reaction (PCR) by using forward primers, reversed primers and probe sequences; and according to difference of Ct values, judging whether threadworm is the xiphinama brevicolle. The method can quickly, accurately and stably identify the xiphinama brevicolle.

Description

technical field [0001] The invention relates to a quarantine method, in particular to a molecular identification method of the short-necked sword nematode. Background technique [0002] From October 2008 to February 2010, a parasitic nematode disease survey was conducted on garden plants in Guangdong Province, and a total of 512 samples were collected for nematode isolation. According to morphological characteristics, morphometric values ​​and binding molecular data, a total of 12 species of 4 genera of potential toxin-transmitting nematodes were identified: a total of 33 samples were found in X. americana group ( Xiphinema americanum group) within the group, in 20 samples were found marked sword nematodes ( X. insigne ) population, X. brasiliensis was found in 27 samples ( X. brasiliense ), accounting for 31.9%, 19.1% and 12.8% of the total number of toxin-transmitting nematode populations detected, respectively. Among them, there is 1 new record species in China: cham...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 张卫东廖力迟远丽徐淼锋
Owner 中华人民共和国珠海出入境检验检疫局
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