Kit for identifying MCR genotyping, identification method and application of kit
A genotyping and kit technology, applied in the fields of medical and health care and microbial detection, can solve the problems of high requirements for instruments and equipment, time-consuming, inefficient, etc., to reduce labor and material consumption, simplify sampling and monitoring, and achieve good specificity. Effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0039] 1. Primer sequence design in the kit of the present invention: by analyzing the MCR gene sequences in all known clinical samples respectively, a total of 59 genes were sequenced using SeqMan ( figure 1 ), find highly conserved segments through analysis, select highly conserved segments with no secondary structure through Premier 3.0, and design multiple sets of primers. The length of primers is generally about 20 bases, and there is no complementarity between primers and within primers sequence. Primer List 1 is as follows:
[0040] Table 1 List of primers and amplified fragment sequences (both 5'-3')
[0041] target gene Primer and probe sequence 5'-3' SEQ ID Amplified fragment length (bp) mcr-1
pre-primer F:5'-CTCGTTGGCTTAGATGACT-3' 1 mcr-1 :167bp
back primer R: 5'-AAGTGCGAACATCAGTCC-3' 2 probe 5'FAM-TCGCTGCCAATAACGGCAAAGATATG-BHQ1 3' 3 mcr-3
pre-primer F:5'-ATATGGGGAGAAAGGAGTTTGAT-3' 4 ...
Embodiment 2
[0060] Select the primer pair described in Table 1 in Example 1, and use the phenol-chloroform method or kits to extract the genomes of bacteria in samples from various sources with the bacterial culture fluid to be checked and the culture fluid of 33 bacterial strains in total of other non-target bacterial strains DNA. Among them, the standard strains were purchased or preserved by the Food Safety and Testing Laboratory of South China University of Technology; the isolates were obtained from the feces of various farms, farmer's markets and healthy people in Shenzhen, and were obtained by using Shen, et al. (2018) method to identify the presence or absence of MCR in the strains, and the strains were all identified by 16S sequencing or mass spectrometry.
[0061] In the 40 μL fluorescent PCR reaction system (prepared according to Table 3), add 2 μL of the genomic DNA of different strains extracted above, and perform fluorescent PCR detection according to the PCR reaction condi...
Embodiment 3
[0074] The feces described in this example are a total of 112 fresh feces samples collected from healthy people using the feces collection kit made by MGI, suspended in the preservation solution, and stored at 4°C for no more than one week.
[0075] Choose the primer pair of table 1 in embodiment 1, will use the total DNA of feces bacteria extracted by the boiling method described in embodiment 1 and adopt the Shen described in embodiment 2, et al The DNA templates extracted by the method of (2018) were used as different DNA templates for PCR detection comparison. The results showed that Shen, et al .(2018) had a positive rate of 12.5% (14 / 112), while this method had a positive rate of 13.4% (15 / 112). It shows that the present invention adopts the boiling method to extract the DNA in the sample to be tested as a template and has a good detection rate.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More