LAMP primer set, kit and detection method of candida auris
A primer set and kit technology, applied in the biological field, can solve problems such as primer-free LAMP detection, and achieve the effects of good specificity, high sensitivity, and high accuracy
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Embodiment 1
[0048] Example 1 designed LAMP primers for Candida auris, and verified the performance of the designed LAMP primers. For common microorganisms, there are a large number of literature reports that can be applied to the detection of genes; but for the research and detection of Candida auris, there are very few, and there is no reported detection gene suitable for Candida auris, which undoubtedly increases the risk of detection of Candida auris. Difficulty of successful primer design for the detection of Candida.
[0049] How to select a detection gene that can be used for Candida auris, first determine whether the gene has the basic conditions as a detection gene, that is, to ensure that the selected gene has a good degree of conservation (high coverage) in this species, It is also necessary to ensure that there is no excessive similarity (high specificity) between the gene and other species. The inventors searched the Candida auris gene sequences entered in the NCBI Gene librar...
Embodiment 2
[0082] Embodiment 2 specificity test
[0083] The first set of LAMP primers was used to perform LAMP detection on the plasmid DNA containing the gene fragment of Candida auris to be detected (SEQ ID NO: 7), one Candida auris strain and six other bacterial species, as shown in Table 2.
[0084] Judge the LAMP reaction results according to the following principles.
[0085] The results judged as a positive reaction were as follows: real-time fluorescent PCR instrument was used for amplification, the reaction time was 50 minutes, and the real-time fluorescence curve and its peak time were observed. Use sterilized purified water as a negative control test, and the result of the negative control test should be negative.
[0086] 1) If Ct≤30, the LAMP test result is determined to be positive;
[0087] 2) If Ct>45, it is determined that the LAMP test result is negative;
[0088] 3) If 30
[008...
Embodiment 3
[0094] Embodiment 3 sensitivity test
[0095] Inoculate the plasmid bacterial liquid containing the gene fragment of Candida auris to be detected (SEQ ID NO: 7) in the LB liquid medium containing ampicillin (concentration ratio: 1:1000), after normal culture for 16 hours, take the ampicillin-containing According to the method of kit instructions, plasmid DNA was extracted (Kangwei century plasmid DNA small extraction kit), and the concentration and purity of plasmid DNA were measured by NanoDrop2000C ultra-micro spectrophotometer. At the same time, the extracted nucleic acid DNA was quantified using the Invitrogen Qubit 2.0 Fluorescence Quantitative Instrument and the Invitrogen Qubit Quantitative Detection Kit, and the copy number was calculated. Finally, use the LAMP test method of the present invention to measure the above DNA samples.
[0096] When the copy number of the plasmid DNA containing the Candida auris gene fragment to be detected is 500 copy / μl, the reaction sho...
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