Kit for detecting carbapenem-resistant Enterobacteriaceae (CRE) and detection method
A technology of enterobacteriaceae and carbapenems, applied in the field of detection of carbapenem-resistant enterobacteriaceae, can solve the problems of high detection cost, low detection throughput, and poor integration level, and achieve simple operation, good effect, and good Effects of Specificity and Efficiency
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Embodiment 1
[0075] Embodiment 1 is used for the preparation of carbapenem-resistant Enterobacteriaceae (CRE) detection kit
[0076] The kit for detecting carbapenem-resistant Enterobacteriaceae (CRE) provided by the present invention consists of the following:
[0077] 1. PCR reaction mixture
[0078] The components and concentrations of the PCR reaction mixture are as follows: 8 μl of Buffer10×buffer solution, pair of detection primers, 0.3 μM of detection probe, and 0.8 mM of each component of dNTP;
[0079] The upstream and downstream detection primers in the detection primer pair are 0.3 μM respectively.
[0080] Detection primer pairs include all of the following detection primer pairs:
[0081] Detection primer pair 1:
[0082] KPC upstream primer is: 5'-ATCGCCGTCTAGTTCTGCTG-3', as shown in SEQ ID NO.1,
[0083] KPC downstream primer is: 5'-TATCCATCGCGTACACACCG-3', as shown in SEQ ID NO.2;
[0084] Detection primer pair 2:
[0085] NDM upstream primer is: 5'-GCAAATGGAAACTGGCGA...
Embodiment 2
[0122] Embodiment 2 is used to detect the use method of carbapenem-resistant Enterobacteriaceae (CRE) kit
[0123] 1. Reaction system preparation
[0124] components
volume
PCR reaction mix
34.4μL
Taq enzyme
0.6μL
sample DNA
5μL
total capacity
40μL
[0125] Take 34.4 μL of PCR reaction mixture, 0.6 μL of Taq enzyme and 5 μL of sample DNA and mix to form 40 μL of reaction solution, vortex and shake evenly, and then centrifuge.
[0126] 2. Fluorescence real-time quantitative PCR reaction and detection
[0127]
[0128] The fluorescent quantitative PCR reaction conditions are shown in the above table: the first stage: 94°C for 5 minutes; the second stage: 94°C for 20s, 58°C for 30s, 40 cycles, and the FAM / HEX signal was collected.
[0129] 3. Result judgment
[0130] After the reaction is over, the amplification curve and cycle number Ct of the samples in each reaction chamber are used as the basis for judging...
Embodiment 3
[0132] Example 3. Sensitivity and specificity analysis for detecting carbapenem-resistant Enterobacteriaceae (CRE) kit
[0133] 1. Preparation of reference DNA nucleic acid
[0134] Construct plasmids containing KPC, NDM, IMP, VIM or OXA-48 genes respectively
[0135] Using KPC, NDM, IMP, VIM or OXA-48 to be tested as templates, each standard product (SEQ ID NO.16-SEQ ID NO.20) as described in the summary of the invention was synthesized by Nanjing GenScript Biotechnology Company. Each standard was connected to the pUC57 vector and sequenced for proofreading.
[0136] The recombinant plasmid containing the KPC gene was named pUC57-PKC; the recombinant plasmid containing the NDM gene was named pUC57-NDM; the recombinant plasmid containing the IMP gene was named pUC57-IMP; the recombinant plasmid containing the VIM gene was named pUC57-VIM; The recombinant plasmid of -48 gene was named pUC57-OXA-48.
[0137] 2. Sensitivity and specificity analysis of the kit for detecting car...
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