Mycoplasma genitalium parC gene mutation type detection method and kit

[0036] Example 1

[0037] 1) Use the sample collection tube provided with the kit to collect patient secretions or urine samples.

[0038] 2) For urine samples, centrifuge at 8,000 rpm for 10 minutes, discard the supernatant, and add an appropriate amount of Lysis buffer to the sample collection tube; for swab samples of secretions, add an appropriate amount of Lysis buffer to the sample collection tube, Stir and soak the swab in Lysis buffer for 5 min.

[0039] 3) The sample collection tube is placed in a metal bath or a water bath, heated at 95° C. for 10 minutes, and allowed to stand at room temperature to complete the extraction of the sample genomic DNA. (The above steps can be used to complete the extraction of genomic DNA with other nucleic acid extraction kits or methods)

[0040] 4) Using the sample genomic DNA obtained in the above steps as a template, 8 mutation types of the parC gene of Mycoplasma genitalium were detected in the primer sets of the two Assays. The 20 μl reaction system includes: 10 μl of EvaGreen Master Mix, the optimal amplification concentration of each primer in the Assay (Table 1), 2 μl of sample genomic DNA, and ddH2O supplemented to 20 μl. The positive control and negative control reaction tubes were simultaneously primers for each detection reaction.

[0041] 5) Amplification reaction and HRM analysis were performed in QuantStudio 6Flex Real-Time PCR System. The conditions of the amplification reaction were: incubation at 95°C for 10 minutes, followed by annealing at 95°C for 15 seconds, and extension at 60°C for 1 minute, for a total of 40 cycles. The temperature was slowly increased at a rate of 95°C and the fluorescence signal was continuously collected.

[0042] 6) After the reaction, use QuantStudio 6and 7Flex Real-Time PCR software v1.0 for analysis, and the software will automatically generate the melting curve and Tm value corresponding to the amplicon. The result interpretation is divided into three steps: in the first step, we need to ensure that all samples are positive for Mycoplasma genitalium (mgpa positive) and confirm that the nucleic acid extraction is successful (HBB positive); in the second step, we then pass the main product in Assay 2 to melt The curve initially identifies the main product type. The main product types are divided into three categories: Type1, Type2, Type3; the third step. The parC genotype was further genotyped by probe peaks amplified by unlabeled probes. The ParC D87 primer set in Assay1 is used to assist in judging the results in the case of impure samples. Notably, since the probe perfectly matched the S83I sequence, the S83I mutant showed a unique peak shape and the highest probe Tm, which allowed us to quickly and directly call S83I. The results were judged by comparing with positive controls of different mutants. (Figure 4).

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