NDM-1 pan-drug resistant gene polymerase chain reaction (PCR) assay kit

[0027] Embodiment 1 This test kit is composed

[0028] The main components are shown in Table 1.

[0029] Table 1

[0030]

[0031] Fluorescence PCR hydrolysis probe method is used. If a single serving is prepared, the components shown in Table 2 are sequentially added to a 0.2mL PCR reaction tube.

[0032] Table 2

[0033] Reagent

Volume (μL)

purified water

27

10×PCR buffer

5

25mM MgCl 2

8

dN(U)TP (10, 20mM)

1

10 μM upstream primer

1

10 μM upstream primer

1

10 μM fluorescent probe

1

Taq enzyme / UNG enzyme system

1

Total

45

[0034] Wherein the sequences of primers and probes are as follows:

[0035] The upstream primer sequence is SEQ ID NO.1: 5'-GCAGGTTGATCTCCTGCTTGAT-3'

[0036] Downstream primer sequence such as SEQ ID NO.2: 5'-GCGTGCTGGTGGTCGATAC-3'

[0037] Fluorescent probe sequence such as SEQ ID NO.3:

[0038] FAM-5'-CAGTTGAGGATCTGG

[0043] Embodiment 2 The use of this kit

[0044] 1. Specimen processing

[0045] Bacterial DNA was extracted using a commercially available bacterial genome extraction kit. The purity A260 / A280 of the extracted DNA is about 1.8.

[0046] 2. PCR amplification

[0047] (1) Take the NDM-1PCR reaction solution, and add 5ul of the sample DNA extraction solution or the supernatant of the quality control product extraction solution, respectively.

[0048] (2) PCR amplification. The PCR reaction tube solution was centrifuged at 6,000rpm, put it into the PCR instrument, and pressed

[0049] The program conditions shown in the following table 3 are amplified:

[0050] table 3

[0051]

[0052] Fluorescein settings: NDM-FAM, TAMRA;

[0053] Fluorescence signal collection: Stage3: 60°C---45sec;

[0054] Reaction volume: 50ul.

[0055](3) Result analysis: automatically save the results after the reaction, and adjust the Start value, End value and Threshold value of the Baseline