Primer-probe combinations for detecting Mycoplasma genitalium drug-resistant genes, their applications and products

Abstract

The present invention provides a primer-probe combination for detecting Mycoplasma genitalium drug-resistant genes, its application and product, belonging to the technical field of biological detection. The present invention provides a primer-probe combination, including primers and a probe for MG 23S rRNA; the primers have nucleotide sequences shown in SEQ ID NO.1-2; the probe has a nucleotide sequence shown in SEQ ID NO.3. The kit prepared from the primer-probe combination provided by the present invention can be used to accurately distinguish mutant samples from wild-type samples. It can be used to detect gene mutations of bacterial 23S rRNA, and a new method for detecting Mycoplasma genitalium macrolide resistance is established.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a primer-probe combination for detecting Mycoplasma genitalium drug-resistant genes, its applications, and products. Background Art

[0002] Macrolide antibiotics have strong antibacterial activities against Gram-positive bacteria and certain Gram-negative bacteria, and also have good effects on microorganisms such as chlamydia and mycoplasma. They are widely used in respiratory and genital tract infectious diseases. In recent years, due to the unreasonable application of a large number of antibiotics in clinical practice, the drug resistance of clinically relevant bacteria has been continuously increasing, leading to the imbalance of normal flora and the emergence of a large number of drug-resistant strains.

[0003] The drug resistance of Mycoplasma genitalium (MG) has developed rapidly. How to control its development and find new drugs for treatment has become an urgent problem to be solved globally. Mycoplasma genitalium infection is the main pathogenic pathogen of male non-chlamydial and non-gonococcal urethritis and is related to female cervicitis and pelvic inflammatory disease (PID). The harm of persistent Mycoplasma genitalium infection and its complications is serious, and untreated Mycoplasma genitalium has the risk of drug-resistant dissemination. Currently, there is no effective vaccine against Mycoplasma genitalium, and effective antibacterial treatment remains the main means for treating and controlling Mycoplasma genitalium infection.

[0004] The macrolide antibiotic azithromycin is the first-line drug for Mycoplasma genitalium infection. However, due to the wide application scenario and high dosage of azithromycin, the drug resistance rate in many Mycoplasma genitalium infections has reached as high as 50%. Given the high macrolide resistance, it is not recommended to use azithromycin without drug resistance detection. Due to the widespread existence of macrolide drug resistance, it is necessary to detect macrolide resistance-mediating mutations (MRMMs) after all Mycoplasma genitalium detections are positive. After performing MRMMs, treatment is carried out according to the results of drug resistance detection, and the cure rate can be increased to over 90%.

[0005] Studies have shown that macrolide resistance in Mycoplasma genitalium is mainly related to gene mutations in bacterial 23S rRNA. Base mutations in the V region of this gene can alter the ribosome structure and prevent drugs from binding to the ribosome. The main mutation sites of the 23S rRNA gene are A2058G, A2059G, and A2058T. In addition, mutations can also occur at the A2058C, A2059C, and A2059T sites. The research by WujianKe et al. in the study named Three Commercial PCR Assays for the Detection of Macrolide Resistance in Mycoplasma genitalium showed that among 101 samples with macrolide resistance mutations, A2059G accounted for 58.42%, A2058G accounted for 20.79%, A2058T accounted for 19.80%, and A2059C accounted for 0.99%.

[0006] Currently, there is no product for detecting macrolide resistance in Mycoplasma genitalium. Provided are a primer-probe combination for detecting drug-resistant genes in Mycoplasma genitalium, its application, and a product. Summary of the Invention

[0007] In view of the above deficiencies, the present invention provides a primer-probe combination for detecting drug-resistant genes in Mycoplasma genitalium, its application, and a product. The present invention provides a primer-probe combination, including primers and a probe for MG 23S rRNA; the primers have nucleotide sequences as shown in SEQ ID NO.1-2; the probe has a nucleotide sequence as shown in SEQ ID NO.3. The kit prepared from the primer-probe combination provided by the present invention can be used to accurately distinguish mutant samples and wild-type samples. It can be used to detect gene mutations in bacterial 23S rRNA, and a new method for detecting macrolide resistance in Mycoplasma genitalium is established.

[0008] The technical solution of the present invention is as follows:

[0009] In the first aspect, the present invention provides a primer-probe combination, and the primer-probe combination includes primers and a probe for MG 23S rRNA.

[0010] Specifically, the primers include a forward primer and a reverse primer, and have nucleotide sequences as shown in SEQ ID NO.1-2.

[0011] Preferably, the forward primer has a nucleotide sequence as shown in SEQ ID NO.1; the reverse primer has a nucleotide sequence as shown in SEQ ID NO.2.

[0012] Specifically, the probe has a nucleotide sequence as shown in SEQ ID NO. 3.

[0013] Preferably, the probe can be labeled with a fluorescent group or a quenching group at the 5'-end or 3'-end.

[0014] Preferably, the fluorescent group is selected from one or more of FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3, CY5, and the quenching group is selected from one or more of DABCYL, BHQ1, BHQ2, BHQ3, ECLIPE.

[0015] Preferably, the probe is labeled with a FAM fluorescent group at the 5'-end and a BHQ1 quenching group at the 3'-end.

[0016] Specifically, the final concentration ratio of the forward primer, reverse primer and probe is 0.2:1:1.

[0017] Preferably, the final concentration of the forward primer is 0.08 μM, the final concentration of the reverse primer is 0.4 μM, and the final concentration of the probe is 0.4 μM.

[0018] In a second aspect, the present invention provides the use of the above primer-probe combination in the preparation of a detection kit for Mycoplasma genitalium drug resistance genes.

[0019] In a third aspect, the present invention provides a kit for detecting Mycoplasma genitalium drug resistance genes, and the kit includes the above primer-probe combination.

[0020] Specifically, the kit further includes a PCR reaction solution, a positive control, and a negative control.

[0021] Preferably, the PCR reaction solution includes the above primer-probe combination, a PCR buffer, a DNA polymerase, dNTP enzymes, dUTP enzymes, MgCl 2 and nuclease-free water.

[0022] Preferably, the positive control is a wild-type target gene plasmid.

[0023] Preferably, the negative control is physiological saline.

[0024] Preferably, the kit is used for detecting Mycoplasma genitalium macrolide resistance.

[0025] In a fourth aspect, the present invention provides a method for detecting Mycoplasma genitalium drug resistance genes, and the method includes using the above kit, and includes the following steps:

[0026] S1. Extract the nucleic acid of the sample to be tested using a nucleic acid extraction kit;

[0027] S2. Prepare the primer-probe combination into a PCR reaction solution;

[0028] S3. Perform PCR amplification detection;

[0029] S4. Result interpretation.

[0030] Preferably, the conditions for the PCR amplification described in step S3 are:

[0031] The first step: 37°C, 2 min;

[0032] The second step: 95°C, 2 min;

[0033] The third step: 95°C, 10 s; 58°C, 20 s; 72°C, 20 s; 45 cycles; collect fluorescence signals at 58°C, 20 s, and the fluorescence channel is FAM;

[0034] The fourth step: 95°C, 1 min; 45°C, 5 min;

[0035] The fifth step: 45 - 90°C, collect fluorescence signals every 0.2°C, keep the temperature constant for 8 s, and the fluorescence channel is FAM.

[0036] Preferably, the criteria for the result interpretation described in step S4 are: if the Tm value of the positive control - 2°C < the Tm value of the sample < the Tm value of the positive control + 2°C, it is determined as a wild-type sample; if 55°C < the Tm value of the sample ≤ the Tm value of the positive control - 2°C, it is determined as a mutant sample.

[0037] The beneficial effects of the present invention are as follows:

[0038] The present invention provides a primer-probe combination, including primers and probes for MG 23S rRNA; the primers have the nucleotide sequences shown in SEQ ID NO.1 - 2; the probes have the nucleotide sequence shown in SEQ ID NO.3. The kit prepared from the primer-probe combination provided by the present invention can be used to accurately distinguish mutant samples and wild-type samples. It can be used to detect gene mutations of bacterial 23S rRNA and establish a new method for detecting macrolide resistance of Mycoplasma genitalium. Brief Description of the Drawings

[0039] Figure 1 Melting peak of wild-type sample (100% wild type).

[0040] Figure 2 Schematic diagram of A2059G result (100% mutant).

[0041] Figure 3 Schematic diagram of A2058G result (100% mutant).

[0042] Figure 4 Schematic diagram of the A2058T result (100% mutant).

[0043] Figure 5 Schematic diagram of the A2059C result (100% mutant).

[0044] Figure 6 Schematic diagram of the A2058C result (100% mutant).

[0045] Figure 7 Melting peak of the 80% mutant + 20% wild-type sample.

[0046] Figure 8 Melting peak of the 70% mutant + 30% wild-type sample.

[0047] Figure 9 Melting peak of the 30% mutant + 70% wild-type sample.

[0048] Figure 10 Melting peak of the 20% mutant + 80% wild-type sample.

[0049] Figure 11 Amplification curve of the serially diluted sample (melting peak of the 20% mutant + 80% wild-type sample).

[0050] Figure 12 Schematic diagram of the melting peak result of the 50 fg sample.

[0051] Figure 13 Schematic diagram of the melting peak result of the 5 fg sample.

[0052] Figure 14 Schematic diagram of the melting peak result of the 0.5 fg sample.

[0053] Figure 15 Melting peak of the sample of Comparative Example 1.

[0054] Figure 16 Melting peak of the sample of Comparative Example 2.

[0055] Figure 17 Melting peak of the sample of Comparative Example 3.

[0056] Figure 18 Melting peak of the sample of Comparative Example 4.

[0057] Figure 19 Melting peak of the sample of Comparative Example 5. Detailed implementation mode

[0058] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0059] Example 1 Primer-probe composition for detecting Mycoplasma genitalium drug-resistant genes

[0060] According to the 23S ribosomal RNA sequence NR_077054.1 in the Mycoplasma genitalium G-37 genomic nucleic acid sequence in the GenBank database, primers and probes were designed using the primer design software Primer Premier 5. The specific methods for primer and probe design are as follows:

[0061] (1) Primer design: At both ends of the detected mutation, the probe covers the mutation site.

[0062] (2) For the MG 23S rRNA probe, the fluorescent group was selected as the common FAM probe labeling dye, and the quenching group was selected as the common BHQ1 probe labeling quencher. The sequence information of the primers and probes is shown in Table 1.

[0063] Table 1 Sequence information of primers and probes

[0064]

[0065] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe; "+" represents Locked Nucleic Acid (LNA) modification.

[0066] Example 2 Kit and usage method for detecting Mycoplasma genitalium drug-resistant genes

[0067] 1. Reagent preparation (solution preparation area)

[0068] (1) First, take out all the reagents from the refrigerator and equilibrate them to room temperature.

[0069] (2) Preparation of the primer-probe mixture: Prepare the primer-probe mixture according to the composition in Table 2.

[0070] Table 2 Composition of the primer-probe mixture

[0071]

[0072] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe.

[0073] (3)Prepare the PCR reaction mixture according to Table 3, add it to a 1.5 mL centrifuge tube, shake well for several seconds, centrifuge at 3000 rpm for several seconds. The prepared PCR reaction mixture can be stored at 4°C for use within 1 h, or stored at -20°C for use within 4 h.

[0074] Table 3 Composition of the PCR reaction mixture

[0075]

[0076] (4)Aliquot the PCR reaction mixture. Aliquot the PCR reaction mixture into PCR thin-walled reaction tubes at 20 μL per tube.

[0077] (5)Transfer the prepared PCR reaction tubes to the extraction room and store them at 4°C or -20°C until the sample extraction and treatment are completed.

[0078] 2. Sample extraction (extraction area)

[0079] (1)DNA extraction of Mycoplasma genitalium samples

[0080] Use nucleic acid extraction or purification reagents to perform steps such as nucleic acid extraction, enrichment, and purification. In the examples of the present invention, MagaBio plus Virus DNA / RNA Purification Kit III (purchased from Bioer Technology, product number BSC86S1B) is used, and the nucleic acid enrichment method is magnetic bead capture method.

[0081] (2)Use a micropipette to add 5 μL of the corresponding DNA extraction sample, negative control, or positive control to each PCR thin-walled reaction tube, and immediately cover the tube lid tightly;

[0082] (3)Transfer the PCR thin-walled reaction tubes with added templates to the PCR amplification area.

[0083] 3. PCR amplification (PCR amplification area)

[0084] (1)The PCR amplification program is set as follows:

[0085] First step: 37°C, 2 min;

[0086] Second step: 95°C, 2 min;

[0087] Third step: 95°C, 10 s; 58°C, 20 s; 72°C, 20 s; 45 cycles; Collect fluorescence signals at 58°C, 20 s, and the fluorescence channel is FAM;

[0088] Fourth step: 95°C, 1 min; 45°C, 5 min;

[0089] Fifth step: 45 - 90°C, collect fluorescence signals every 0.2°C, keep the temperature constant for 8 s, and the fluorescence channel is FAM.

[0090] (2) After the program runs to completion, take out the PCR thin-walled reaction tube and place it in a concave-convex bag. Seal the bag tightly and handle it according to the pollution source.

[0091] 4. Result determination

[0092] Result determination of macrolide resistance mutations in Mycoplasma genitalium: After the PCR reaction is completed, the product is analyzed by melting curve. According to the differences in the melting points and shapes of the melting peaks, the wild type and mutant types are distinguished, that is, the detection of macrolide resistance mutations in Mycoplasma genitalium is completed.

[0093] The specific method for distinguishing the wild type and mutant types is as follows:

[0094] If the Tm value of the sample satisfies positive control Tm value - 2°C < sample Tm value < positive control Tm value + 2°C, it is determined as a wild-type sample; if 55°C < sample Tm value ≤ positive control Tm value - 2°C, it is determined as a mutant sample.

[0095] 5. Reference values for drug resistance gene mutation detection

[0096] Positive control, that is, the Tm value ranges of the wild type in each system and each channel are as follows: The Tm value of the wild-type control peak in the FAM channel of reaction system A is 74.8°C ( Figure 1 )

[0097] Among them, each Tm value is a common value obtained on a specific Hongshi SLAN-96S instrument. As a reference, when using other instruments, the Tm value may vary slightly, and the Tm value obtained from the positive control of the current test shall prevail;

[0098] The Tm value is subject to the automatic interpretation of the instrument. When the instrument gives more than one Tm value, please refer to the peak shapes of the positive control and negative control to select the valid Tm value; when the instrument cannot automatically give the Tm value, obtain the Tm value by adjusting the baseline or directly through manual interpretation.

[0099] Example 3 Use of the kit for detecting different drug resistance mutation types of Mycoplasma genitalium

[0100] The mutant sample is the MG 23S rRNA mutant plasmid, synthesized by Shanghai Jierui Biotechnology Co., Ltd., including A2059G, A2058G, A2058T, A2059C, and A2058C, a total of five mutant plasmids. Each mutant plasmid is independently synthesized and quantified.

[0101] Dilute each plasmid to an appropriate concentration with TE buffer. The samples are measured using the kit described in Example 2 with reference to the method described in Example 2. The schematic diagram of the A2059G result is as Figure 2 shown, and the schematic diagram of the A2058G result is asFigure 3 As shown in Figure 4 As shown in Figure 5 As shown in Figure 6 As shown. The results show that the kit for detecting Mycoplasma genitalium drug resistance genes of the present invention can accurately distinguish mutant samples and wild-type samples. It can be used to detect gene mutations of bacterial 23S rRNA and for detecting Mycoplasma genitalium macrolide resistance.

[0102] Example 4 Use of the kit for detecting different drug resistance mutation ratios of Mycoplasma genitalium

[0103] The samples were measured using the kit described in Example 2 with reference to the method described in Example 2.

[0104] The wild-type sample is the wild-type plasmid of MG 23S rRNA, synthesized by Shanghai Jierui Biotechnology Co., Ltd. The mutant sample is the mutant plasmid of MG 23S rRNA, synthesized by Shanghai Jierui Biotechnology Co., Ltd. Each mutant plasmid was independently synthesized and quantified.

[0105] In this example, taking the A2059G mutation type as an example, each plasmid was diluted to the same concentration with TE buffer to prepare samples to be measured with different drug resistance mutation ratios. The method is shown in Table 4 below:

[0106] Table 4 Preparation of samples with different mutation ratios

[0107]

[0108] The samples were measured using the kit described in Example 2 with reference to the method described in Example 2.

[0109] The schematic diagram of the results of the 80% mutant + 20% wild-type sample is as shown in Figure 7 As shown; the schematic diagram of the results of the 70% mutant + 30% wild-type sample is as shown in Figure 8 As shown; the schematic diagram of the results of the 30% mutant + 70% wild-type sample is as shown in Figure 9 As shown; the schematic diagram of the results of the 20% mutant + 80% wild-type sample is as shown in Figure 10 As shown. The results show that the kit for detecting Mycoplasma genitalium drug resistance genes of the present invention can accurately distinguish mutant samples and wild-type samples. It can be used to detect gene mutations of bacterial 23S rRNA and for detecting Mycoplasma genitalium macrolide resistance.

[0110] Example 5 Sensitivity of the kit for detecting Mycoplasma genitalium drug resistance genes

[0111] The wild-type sample is the wild-type plasmid of MG 23S rRNA, which was synthesized by Shanghai Jierui Biotechnology Co., Ltd. The mutant sample is the mutant plasmid of MG 23S rRNA, which was synthesized by Shanghai Jierui Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently.

[0112] The wild-type and mutant plasmids were diluted in a 10-fold gradient using TE buffer to 10 fg / μL, 1 fg / μL, and 0.1 fg / μL, respectively. Subsequently, wild-type and mutant plasmids of the same concentration were mixed to a 20% mutation ratio. The sample mixing method is shown in Table 5 below:

[0113] Table 5 Preparation of Sensitivity Detection Samples (20% Mutation Ratio)

[0114]

[0115] Samples of each concentration gradient were prepared according to Table 5. The samples were measured using the kit described in Example 2 with reference to the method described in Example 2. The sample addition volume was 5 μL, so the total plasmid sample addition amounts were 50 fg, 5 fg, and 0.5 fg, respectively.

[0116] The schematic diagram of the amplification curve results of samples with different concentration gradients is as Figure 11 shown, and the schematic diagram of the melting peak map results of the 50 fg sample is as Figure 12 shown, the schematic diagram of the melting peak map results of the 5 fg sample is as Figure 13 shown, and the schematic diagram of the melting peak map results of the 0.5 fg sample is as Figure 14 shown. The results show that the kit for detecting Mycoplasma genitalium drug resistance genes of the present invention can accurately distinguish mutant samples and wild-type samples. It can be used to detect gene mutations of bacterial 23S rRNA and for Mycoplasma genitalium macrolide resistance detection.

[0117] Comparative Example 1 Kits Containing Different Primer-Probe Mixtures

[0118] The difference between Comparative Example 1 and Example 2 is only that: the composition of the primer-probe mixture is different. The primer-probe mixture was prepared according to the composition in Table 6.

[0119] Table 6 Primer-Probe Mixture of Comparative Example 1

[0120]

[0121] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe.

[0122] Comparative Example 2 Kits Containing Different Primer-Probe Mixtures

[0123] The difference between Comparative Example 2 and Example 2 is only that: the composition of the primer-probe mixture is different. Prepare the primer-probe mixture according to the composition in Table 7.

[0124] Table 7 Primer-probe mixture of Comparative Example 2

[0125]

[0126] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe; "+" represents the Locked Nucleic Acid (LNA) modification.

[0127] Comparative Example 3 Kit containing different primer-probe mixtures

[0128] The difference between Comparative Example 3 and Example 2 is only that: the composition of the primer-probe mixture is different. Prepare the primer-probe mixture according to the composition in Table 8.

[0129] Table 8 Primer-probe mixture of Comparative Example 3

[0130]

[0131] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe; "+" represents the Locked Nucleic Acid (LNA) modification.

[0132] Comparative Example 4 Kit containing different primer-probe mixtures

[0133] The difference between Comparative Example 4 and Example 2 is only that: the composition of the primer-probe mixture is different. Prepare the primer-probe mixture according to the composition in Table 9.

[0134] Table 9 Primer-probe mixture of Comparative Example 4

[0135]

[0136] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; "P" represents the probe; "+" represents the Locked Nucleic Acid (LNA) modification.

[0137] Comparative Example 5 Kit containing different primer-probe mixtures

[0138] The difference between Comparative Example 5 and Example 2 is only that: the composition of the primer-probe mixture is different. Prepare the primer-probe mixture according to the composition in Table 10.

[0139] Table 10 Primer-probe mixture of Comparative Example 5

[0140]

[0141] Experimental Example 1

[0142] Using the primer-probe mixture of Comparative Examples 1-5, a kit was prepared according to the method described in Example 2 to test the samples. The samples used for the test were the "30% mutant + 70% wild-type" samples in Example 4.

[0143] The detection result of Comparative Example 1 is as Figure 15 shown, the baseline of the melting peak is uneven; the detection result of Comparative Example 2 is as Figure 16 shown, the baseline of the melting peak is uneven and the positions of the wild-type peak and the mutant peak cannot be judged; the detection result of Comparative Example 3 is as Figure 17 shown, there is only a single melting peak and the Tm value is relatively low. The test result of Comparative Example 4 is as Figure 18 shown, there is no obvious melting peak. The schematic diagram of the detection result of Comparative Example 5 is as Figure 19 shown, the position of the wild-type melting peak is accurate, but the peak height of the mutant melting peak is reduced. When using the software to unify the interpretation rules, the mutant melting peak cannot be accurately identified, which is likely to cause some mutant samples to be missed.

[0144] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A primer-probe combination, characterized in that: The primer-probe combination consists of primers and probes of MG 23S rRNA; The primers are composed of a forward primer and a reverse primer, and the nucleotide sequence is shown in SEQ ID NO.1-2; the nucleotide sequence of the probe is shown in SEQ ID NO.3; The nucleotide sequence of the probe is modified with locked nucleic acid at the 6th, 7th and 8th bases at the 5' end; The probe is labeled with a fluorescent group at the 5' end, which is FAM; and a quenching group at the 3' end, which is BHQ1; The final concentration ratio of the forward primer, reverse primer and probe is 0.2:1:

1.

2. The primer-probe combination according to claim 1, characterized in that: The forward primer has a nucleotide sequence as shown in SEQ ID NO.1; the reverse primer has a nucleotide sequence as shown in SEQ ID NO.

2.

3. Use of the primer-probe combination according to any one of claims 1 to 2 in the preparation of a kit for detecting drug-resistant genes of Mycoplasma genitalium.

4. A kit for detecting drug-resistant genes of Mycoplasma genitalium, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 2.

5. The kit according to claim 4, characterized in that The kit also includes PCR reaction solution, positive control substance and negative control substance.

6. The kit according to claim 5, characterized in that The negative control substance is physiological saline; the positive control substance is a wild-type target gene plasmid.

7. A method for detecting Mycoplasma genitalium resistance genes, characterized in that: The method comprises using the kit according to any one of claims 5 to 6, and the method is a method for non-diagnostic purposes, comprising the following steps: S1. Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested; S2, preparing the primer-probe combination into a PCR reaction solution; S3, performing PCR amplification detection; S4. Interpretation of results.

8. The method according to claim 7, characterized in that The standard for interpreting the results in step S4 is: if the positive control Tm value -2°C < sample Tm value < positive control Tm value +2°C, it is determined to be a wild-type sample; if 55°C < sample Tm value ≤ positive control Tm value -2°C, it is determined to be a mutant sample.

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