Method for detecting radopholus similis by real-time fluorescent polymerase chain reaction (PCR)
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A technology of real-time fluorescence of banana perforator nematodes, applied in the fields of fluorescence/phosphorescence, biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve the problem that larvae and males cannot be quarantined, detected and identified, and the accuracy rate is not high. cost and other issues, to achieve the effect of saving quarantine, avoiding pollution, and high sensitivity
Inactive Publication Date: 2011-08-31
SHENZHEN ACAD OF INSPECTION & QUARANTINE +1
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The morphological method requires relevant personnel to have rich experience, and because of the intraspecific variation of the banana borer nematode, the accuracy of the quarantine, detection and identification results of this method is often not high, and the time required is related to the experience of the operator; The isoenzyme method can only carry out quarantine, detection and identification of females, but cannot carry out quarantine, detection and identification of larvae and males, and the time required is as long as about one day; the molecular diagnosis method based on PCR can basically meet the requirements of banana perforator nematodes. Accurate and rapid quarantine, detection and identification requirements, according to relevant literature reports, the current quarantine, detection and identification method using a pair of common PCR primer amplification still needs more than 2 hours, while the quarantine, detection and identification method using double PCR primer amplification and identification method, although it only takes 54 minutes, a pair of primers is added, which increases the cost of quarantine, detection and identification
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specific Embodiment approach 1
[0038] A method for real-time fluorescent polymerase chain reaction detection of a large number of banana nematodes, which has the following steps in turn:
[0039] 1) Synthetic probes and primers
[0040] First select the probe RsP, then design a pair of specific primers consisting of a specific forward primer Rsf-F and a specific reverse primer Rsf-R as close as possible to the probe, and then according to the probe RsP and specificity The sequences of the forward primer Rsf-F and the specific reverse primer Rsf-R are respectively synthesized as probes and specific primers for use;
[0041] The sequence of the probe RsP is:
[0042] 5'-FAM-AGCGGTTGTAGTCCAT-MGB-3';
[0043] The sequence of the forward primer Rsf-F is:
[0044] 5'-CTGGGTTGGCGTCTGTGAGT-3';
[0045] The sequence of the reverse primer Rsf-R is:
[0046] 5'-CGACCTACGAGCCGAGTGA-3';
[0047] 2) Cultivate nematodes
[0048] Cultivate the banana perforator colony on the carrot slices, and also cultivate the ban...
specific Embodiment approach 2
[0060] A method for real-time fluorescent polymerase chain reaction detection of a single banana perforator nematode, which has the following steps in turn:
[0061] 1) Synthetic probes and primers
[0062] First select the probe RsP, then design a pair of specific primers consisting of a specific forward primer Rsf-F and a specific reverse primer Rsf-R as close as possible to the probe, and then according to the probe RsP and specificity The sequences of the forward primer Rsf-F and the specific reverse primer Rsf-R are respectively synthesized as probes and specific primers for use;
[0063] The sequence of the probe RsP is:
[0064] 5'-FAM-AGCGGTTGTAGTCCAT-MGB-3';
[0065] The sequence of the forward primer Rsf-F is:
[0066] 5'-CTGGGTTGGCGTCTGTGAGT-3';
[0067] The sequence of the reverse primer Rsf-R is:
[0068] 5'-CGACCTACGAGCCGAGTGA-3';
[0069] 2) Cultivate nematodes
[0070] Cultivation of a banana borer nematode colony on carrot slices;
[0071] 3) Extractin...
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Abstract
The invention relates to a method for detecting radopholus similis by real-time fluorescent polymerasechain reaction (PCR). The sequence of a probe is 5'-FAM-AGCGGTTGTAGTCCAT-MGB-3'; the sequence of a forward primer is 5'-CTGGGTTGGCGTCTGTGAGT-3'; and the sequence of a reverse primer is 5'-CGACCTACGAGCCGAGTGA-3'. The extraction method for nucleic acid of radopholus similis has high extraction efficiency, and the purity of the extracted nucleic acid is high. A real-time fluorescent PCRamplifier can be used for quickly and accurately quarantining, detecting and identifying larvae, female wormsand male worms of radopholus similis and even larvae, female worms and male worms of single radopholus similis. The reaction time is only 45 minutes, the detection result is directly displayed on a computer screen without electrophoresis, the time is saved, and the pollution is prevented. In addition, the amplifying sensitivity is high, wherein 1 / 40 of the DNA content of single radopholus similiscan be used as a DNA sample template for PCR amplifying reaction.
Description
technical field [0001] The invention relates to an assay or inspection method comprising enzymes or microorganisms, in particular to a real-time fluorescent polymerasechain reaction method for detecting the banana borer nematode. Background technique [0002] Banana punch nematode (Radopholus similis Thorne, 1949), also known as banana root rot, is an obligate parasitic nematode that harms bananas, flower planting and many commercial crops. It has a wide range of hosts and causes great economic losses. It belongs to sovereign countries Phytosanitary hazardous pests prohibited from entry. In order to effectively prevent the entry of banana borer nematodes, the commonly used existing quarantine, detection and identification methods are morphological methods, isozyme methods and polymerasechain reaction (PolymeraseChain Reaction, referred to as PCR) methods. The morphological method requires relevant personnel to have rich experience, and because of the intraspecific variat...
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