PLRV (Potato Leafroll Virus) RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification) detection kit and PLRV RT-LAMP detection method
An RT-LAMP and detection kit technology, applied in the field of microbial detection, can solve the problems of inability to detect dormant seed potatoes, difficult to detect viruses, etc., and achieve the effects of low cost, simple identification and low false positive rate.
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Embodiment 1
[0019] The preparation of embodiment 1 potato leafroll virus RT-LAMP detection kit
[0020] 1.1 Reagents
[0021] Primers were synthesized by Tiangen Biochemical Technology (Beijing) Co., Ltd.; BstDNA polymerase and 10×ThermoPol reaction buffer were purchased from NEB; AMV reverse transcriptase was purchased from Promega; SYBRGreenI and TRIZOLReagent were purchased from Invitrogen; other PCR reagents were purchased from Sigma.
[0022] 1.2 Preparation of the kit:
[0023] Reaction buffer, prepared according to the following formula: 2mM dNTP, 10×ThermoPol reaction buffer, 0.6mM betaine and 6mMMg 2+ ;
[0024] 4 specific primers: outer primer F3, its nucleotide sequence is as shown in SEQIDNO:1; Outer primer B3, its nucleotide sequence is as shown in SEQIDNO:2; Internal primer FIP, its nucleotide sequence is as shown in SEQIDNO: Shown in 3, internal primer BIP, its nucleotide sequence is shown in SEQIDNO:4. The concentration of outer primer F3 and outer primer B3 is 0.2 μm...
Embodiment 2
[0030] Example 2 Potato leafroll virus specific detection
[0031] 2.1 RT-LAMP specific detection
[0032] The samples to be tested were potato leaves collected from Hebei Yongnian Vegetable Base, including 6 samples infected with PLRV identified by ELISA and 2 samples each infected with Potato virus X, Potato virus Y, Potato virus A and Cucumber mosaic virus .
[0033] Use the kit prepared in Example 1 to detect the above samples according to the following steps:
[0034] 1. Use TRIZOLReagent to extract the RNA of the sample to be tested, the specific method is as follows:
[0035] (1) Cut off an appropriate amount of leaves, fully grind, add 1ml TrizolReagent to fully grind, and place at room temperature for 5 minutes;
[0036] (2) Add 200 μl of chloroform, shake fully for 15 sec, leave at room temperature for 2-3 min, then centrifuge at 12000×g, 4°C for 10 min;
[0037] (3) Take the upper aqueous phase into a new tube, add an equal volume of isopropanol, place at room t...
Embodiment 3
[0045] Example 3 Sensitivity Detection of Potato Leafroll Virus
[0046] 3.1 Sensitivity detection of RT-LAMP
[0047]The RNA samples identified as Potato leafroll virus positive in Example 2 were taken and diluted into 7 gradients of 100fg-100ng by a 10-fold concentration serial dilution method.
[0048] The RT-LAMP reaction and result interpretation steps are the same as steps 2-4 in Example 2.
[0049] 3.2 Test results
[0050] Except that the PCR tube with a DNA concentration of 100fg shows orange, the PCR tubes with other concentrations all show green, indicating that the minimum detection limit of the detection method of the present invention reaches 1pgDNA, and the sensitivity is very high.
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