CN115198006A - An XNA molecular clamp-multiplex fluorescent PCR method for gene mutation detection - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to an XNA molecular clamp-multiplex fluorescence PCR method for gene mutation detection; comprising preparing a sample to be tested; extracting DNA and RNA in the sample to be tested, and reversing the RNA, The cRNA is obtained, and then the cRNA and the DNA are mixed and subjected to multiple fluorescent PCR diffusion to obtain a fluorescent sample; the fluorescent sample is detected by a fluorescent PCR amplifier, and the ct value of the fluorescent sample is displayed to determine the detection result. Methods The multiplex fluorescence PCR diffusion technology was introduced, and the real-time PCR system was established by using specific primer and probe technology and three-color fluorescence channel detection mode, which could detect multiple fusion genes and multiple mutant genes at the same time, with good specificity and accuracy. It has high rate, fast and cheap, and solves the problem of time-consuming detection of gene mutation status by existing kits.

Description

An XNA molecular clamp-multiplex fluorescent PCR method for gene mutation detection

technical field

The invention relates to the technical field of microorganisms, in particular to an XNA molecular clamp-multiplex fluorescence PCR method for gene mutation detection.

Background technique

Lung cancer is one of the common malignant tumors that seriously endanger human health, and 80-85% of lung cancers are non-small cell lung cancers. There are various types of gene mutation in patients with non-small cell lung cancer, among which the mutation frequency of EGFR, HER2, KRAS, BRAF gene is about 10-50%, 1-4%, 5-25%, 1-2%, ALK, ROS1 , RET, NTRK1, NTRK2, NTRK3 gene fusion frequency is about 3-7%, 1%, 1%, 0.12%, 0.02%, 0.08%, MET exon 14 skip gene mutation frequency is about 1%. A large number of clinical studies have shown that the driver gene mutation status is an important predictor of the efficacy of targeted drug therapy, and it is necessary to detect the gene mutation status before targeted therapy. Combined detection of multiple gene mutations in patients with non-small cell lung cancer can provide patients with more precise treatment.

XNA molecular clamp is a novel nucleic acid molecule oligomer that hybridizes to target DNA sequences and can be used as a molecular clamp in real-time quantitative polymerase chain reaction. The XNA molecular clamp assay can produce highly sensitive results using liquid biopsy and FFPE samples.

These gene mutations or fusion states are related to the efficacy of targeted drugs. However, most of the current kits can only detect one or several gene mutations at a time. The detection throughput is low, time-consuming and requires a large amount of samples.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an XNA molecular clamp-multiplex fluorescent PCR method for gene mutation detection method, which solves the problem that the detection of gene mutation status takes a long time in the existing kits.

To achieve the above purpose, the present invention provides an XNA molecular clamp-multiplex fluorescent PCR method for gene mutation detection:

prepare samples to be tested;

Extracting the DNA and RNA in the sample to be tested, and reversing the RNA to obtain cRNA, then mixing the cRNA and the DNA and performing multiple fluorescence PCR diffusion to obtain a fluorescent sample;

The fluorescent sample is detected by a fluorescent PCR amplifier, and the ct value of the fluorescent sample is displayed to determine the detection result.

Wherein, the specific way of preparing the sample to be tested:

All saliva and fecal samples from healthy people with no history of lung disease were inoculated into the culture medium, and cultured at 37°C for 20-40h anaerobic;

Take 4ml of the culture solution in each of the culture medium and centrifuge, 5500rpm, 10min, the thalline after centrifugation is washed with sterile physiological saline with pH 6.8, and centrifuge again after washing, 5500rpm, 10min to obtain sterile pure water suspended bacteria;

Suspend the bacterial cells in 400 uL of the sterile pure water, take a boiling water bath for 10 minutes, centrifuge at 10,000 rpm for 15 minutes, and collect the supernatant.

The specific method of extracting the DNA and RNA in the sample to be tested, and reversing the RNA to obtain cRNA, and then mixing the cRNA and the DNA to perform multiple fluorescence PCR diffusion to obtain a fluorescent sample:

Extracting DNA and RNA in the sample to be tested, heating the extracted RNA into a reverse transcription reaction solution, and mixing the RNA with the reverse transcriptase in the reverse transcription reaction solution to obtain cRNA;

The DNA and the cRNA are respectively mixed with a mixed enzyme, and the mixed DNA and the cRNA are subjected to multiplex fluorescence PCR diffusion to obtain the fluorescent sample.

The reaction conditions of the PCR amplification were pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10S, annealing at 55°C for 30S, extension at 72°C for 90S, the entire system was performed for 28 cycles, and extension at 72°C for 5 min.

Wherein, the fluorescent PCR amplifier includes a casing, an amplifier body, a temperature regulation plate and a placement component, the amplifier body is fixedly connected to the casing, and is located in the casing, and the temperature regulation The plate is arranged in the casing, and the placing component is arranged on the side of the temperature regulation plate away from the body of the amplification instrument.

Wherein, the placing assembly includes a placing plate, a push rod, a fixing rod and a slider, the push rod is disposed on the side of the casing close to the amplification instrument body, and the placing plate is slidably connected to the casing , and is located in the housing, the housing has a slot, the placement plate has a chute, the fixing rod is arranged in the chute, the slider is fixedly connected with the fixing rod, and penetrates through the the placement plate.

Wherein, the fixing rod includes a sliding rod and a first spring, the sliding rod is slidably connected with the placing plate and is located in the sliding groove, the first spring is fixedly connected with the sliding rod, and is connected with the The placing plate is fixedly connected and located between the sliding rod and the placing plate.

A method for detecting gene mutation by XNA molecular clamp-multiplex fluorescence PCR method of the present invention prepares a sample to be tested; extracts DNA and RNA in the sample to be tested, and reverses the RNA to obtain cRNA, and then the After the cRNA and the DNA are mixed, multiplex fluorescent PCR diffusion is performed to obtain a fluorescent sample; the fluorescent sample is detected by a fluorescent PCR amplifier, and the ct value of the fluorescent sample is displayed to determine the detection result. This method introduces the multiple fluorescent PCR diffusion technology , By using specific primers and probe technology, three-color fluorescence channel detection mode, a real-time PCR system has been established, which can detect multiple fusion genes and multiple mutant genes at the same time, with good specificity, high accuracy, fast and cheap, and the solution The detection of gene mutation status by existing kits is time-consuming.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

FIG. 1 is a flow chart of a method for detecting gene mutation by XNA molecular clamp-multiplex fluorescence PCR method provided by the present invention.

FIG. 2 is a schematic structural diagram of the fluorescence PCR amplifier provided by the present invention.

FIG. 3 is an internal cross-sectional view of the fluorescence PCR amplifier provided by the present invention.

In the picture: 1-shell, 2-card slot, 3-amplifier body, 4-thermostat plate, 5-placement plate, 6-chute, 7-slide bar, 8-first spring, 9-slider Block body, 10-draw rod, 11- telescopic rod, 12-second spring.

Detailed ways

The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention and should not be construed as limiting the present invention.

Please refer to FIG. 1 to FIG. 3 , the present invention provides an XNA molecular clamp-multiplex fluorescence PCR method for gene mutation detection, comprising the following steps:

S1 prepares the sample to be tested;

Specific way:

S11 Inoculate saliva and feces samples from healthy people with no history of lung disease into culture medium, and inoculate anaerobic culture at 37°C for 20-40h;

S12 respectively take 4 ml of the culture liquid in each of the medium and centrifuge, 5500rpm, 10min, after the centrifugation, the cells are washed with sterile physiological saline with pH 6.8, and centrifuged again after washing, 5500rpm, 10min, to obtain sterile pure water suspension bacteria;

S13 Suspend the bacterial cells in 400 uL of the sterile pure water, take a boiling water bath for 10 minutes, centrifuge at 10,000 rpm for 15 minutes, and collect the supernatant.

S2 extracts the DNA and RNA in the sample to be tested, and reverses the RNA to obtain cRNA, and then mixes the cRNA and the DNA and performs multiple fluorescence PCR diffusion to obtain a fluorescent sample;

Specifically, the reaction conditions of the PCR amplification are as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10S, annealing at 55°C for 30S, extension at 72°C for 90S, the whole system is performed for 28 cycles, and extension at 72°C for 5 min. The amplification reaction system is: 2×TaqPCR mix 10 μL, DDH ₂ O 9 μL, upstream primer F1 0.4 μL, downstream primer R1 0.4 μL and sample template 0.2 μL, vortex mixing, flash off for 5 s, and place on PCR nucleic acid amplifier for reaction .

Specific way:

S21 extracts DNA and RNA in the test sample, heats the extracted RNA into a reverse transcription reaction solution, and mixes the RNA with the reverse transcriptase in the reverse transcription reaction solution to obtain cRNA;

S22 The DNA and the cRNA are mixed with a mixed enzyme, respectively, and the mixed DNA and the cRNA are subjected to multiplex fluorescence PCR diffusion to obtain the fluorescent sample.

S3 detects the fluorescent sample by a fluorescent PCR amplifier, and the displayed ct value determines the detection result.

Specifically, specific primers and probes are used for PCR reaction, the mutation is indicated by the FAM/ROX/CY5 signal, and the internal control is indicated by the VIC signal. The Δct value of the reaction tube is judged by calculating the Δct value ≤ cut off value. When the sample is positive for the corresponding signal mutation of the reaction tube, it is negative or lower than the detection limit of the kit. Δct value = ct value mutation site - ct value internal control.

Further, the fluorescent PCR amplification instrument includes a housing 1, a amplification instrument body 3, a temperature regulating plate 4 and a placement component, and the amplification instrument body 3 is fixedly connected with the housing 1 and is located in the housing. Inside the body 1 , the temperature regulating plate 4 is arranged in the casing 1 , and the placing component is arranged on the side of the temperature regulating plate 4 away from the amplification instrument body 3 ; the placing component includes a placing plate 5 , a mandrel, a fixing rod and a slider, the mandrel is arranged on the side of the housing 1 close to the amplification instrument body 3, the placing plate 5 is slidably connected with the housing 1, and is located in the Inside the casing 1, the casing 1 has a card slot 2, the placing plate 5 has a sliding groove 6, the fixing rod is arranged in the sliding groove 6, and the sliding block is fixedly connected with the fixing rod, and pass through the placing plate 5; the fixing rod includes a sliding rod 7 and a first spring 8, the sliding rod 7 is slidably connected with the placing plate 5, and is located in the sliding groove 6, the first spring 8 is fixedly connected with the sliding bar 7 and fixedly connected with the placing plate 5 , and is located between the sliding bar 7 and the placing plate 5 .

In this embodiment, the housing 1 provides installation conditions for the thermal cycler body 3 , the temperature regulation plate 4 and the placement assembly, and the thermal cycler body 3 uses polymerase chain reaction technology to The DNA and the cRNA are amplified, the temperature regulating plate 4 is connected to a cooling liquid source and a hot water source, and the test tube containing the DNA and the cRNA is heated and cooled, and the placement plate 5 is used to place the device. The test tube with the DNA and the cRNA can be placed in multiple test tubes at the same time for detection. When placing the test tube in the housing 1, the test tube is placed in the placement plate 5, and the inspector uses his hand to pull it to the middle The sliding block, the sliding block slides the fixing rod into the placing plate 5, the inspector puts the placing plate 5 into the casing 1, loosens the sliding block and lowers it down. Press the placing plate 5 so that the placing plate 5 slides in the housing 1 and squeezes the ejector rod. When the sliding slot 6 and the card slot 2 are at the same level, the first spring 8 Push the slide bar 7 to slide out of the chute 6, and insert the slide bar 7 into the card slot 2 to fix the placing plate 5. When taking out the test tube from the housing 1, the inspector Slide the slider to the middle again, the slider slides so that the sliding rod 7 retracts into the chute 6, separates the sliding rod 7 from the housing 1, and the top rod is reset to remove the The placing plate 5 is lifted up, which is convenient for the inspector to take out the placing plate 5 from the housing 1 .

Further, the slider includes a slider body 9 and a pull rod 10 . The slider body 9 is fixedly connected to the slider rod 7 and penetrates through the placing plate 5 . The pull rod 10 is connected to the slider body 9 . It is fixedly connected and located on one side of the slider body 9 .

In this embodiment, the setting of the pull rod 10 is convenient for the inspector to slide the slider body 9 , and the slider body 9 slides to control the sliding of the sliding rod 7 .

Further, the top rod includes a telescopic rod 11 and a second spring 12. The telescopic rod 11 is fixedly connected to the housing 1 and is located on the side close to the amplification instrument body 3. The second spring 12 It is sleeved on the telescopic rod 11 .

In this embodiment, when the placing plate 5 is pressed down, the placing plate 5 pushes the telescopic rod 11 so that the telescopic rod 11 and the second spring 12 are compressed, and when the test tube is taken out, the sliding rod 7 is separated from the housing 1, and the second spring 12 resets and drives the telescopic rod 11 to extend, so as to lift the placing plate 5.

The above disclosure is only a preferred embodiment of the present invention, and of course, it cannot limit the scope of rights of the present invention. Those of ordinary skill in the art can understand that all or part of the process for realizing the above-mentioned embodiment can be realized according to the rights of the present invention. The equivalent changes required to be made still belong to the scope covered by the invention.

Claims (7)

1. A gene mutation detection method by an XNA molecular clamp-multiplex fluorescence PCR method is characterized by comprising the following steps:

preparing a sample to be detected;

extracting DNA and RNA in the sample to be detected, reversing the RNA to obtain cRNA, mixing the cRNA with the DNA, and performing multiple fluorescence PCR diffusion to obtain a fluorescence sample;

and detecting the fluorescent sample by a fluorescent PCR (polymerase chain reaction) amplification instrument, and displaying the ct value of the fluorescent sample to judge the detection result.

2. The method of claim 1, wherein the gene mutation is detected by the XNA molecular clamp-multiplex fluorescence PCR method,

the specific mode for preparing the sample to be detected is as follows:

inoculating saliva and feces samples of healthy people with no lung disease history into a culture medium, standing at 37 ℃ and carrying out anaerobic culture for 20-40h;

centrifuging 4ml of culture solution in each culture medium at 5500rpm for 10min, washing the centrifuged thallus with sterile physiological saline with the pH of 6.8, centrifuging again at 5500rpm for 10min to obtain sterile pure water suspended thallus;

400uL of the sterile pure water was suspended in the cells, centrifuged in boiling water bath for 10min and 10000rpm for 15min, and the supernatant was collected.

3. The method for detecting gene mutation by XNA molecular clamp-multiplex fluorescence PCR method as claimed in claim 1,

the specific mode of extracting DNA and RNA in the sample to be detected, reversing the RNA to obtain cRNA, mixing the cRNA with the DNA, and performing multiple fluorescence PCR diffusion to obtain a fluorescence sample is as follows:

extracting DNA and RNA in the sample to be detected, heating the extracted RNA into a reverse transcription reaction solution, and mixing the RNA with reverse transcriptase in the reverse transcription reaction solution to obtain cRNA;

and respectively mixing the DNA and the cRNA with mixed enzyme, and performing multiplex fluorescence PCR diffusion on the mixed DNA and the mixed cRNA to obtain the fluorescence sample.

4. The method of claim 1, wherein the gene mutation is detected by the XNA molecular clamp-multiplex fluorescence PCR method,

the reaction conditions of PCR amplification are pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 10S, annealing at 55 ℃ for 30S and extension at 72 ℃ for 90S, the whole system is subjected to 28 cycles, and extension is carried out for 5min at 72 ℃.

5. The method of claim 1, wherein the gene mutation is detected by the XNA molecular clamp-multiplex fluorescence PCR method,

fluorescence PCR amplifys appearance includes casing, amplifys appearance body, temperature regulating plate and places the subassembly, the amplifys appearance body with casing fixed connection, and be located in the casing, the temperature regulating plate set up in the casing, place the subassembly set up in the temperature regulating plate is kept away from amplifys appearance body one side.

6. The method of claim 5, wherein the gene mutation is detected by multiplex fluorescence PCR using molecular tweezer-multiplex fluorescence PCR,

the placing assembly comprises a placing plate, an ejector rod, a fixed rod and a sliding block, the ejector rod is arranged on one side, close to the amplification instrument body, of the shell, the placing plate is connected with the shell in a sliding mode and located in the shell, the shell is provided with a clamping groove, the placing plate is provided with a sliding groove, the fixed rod is arranged in the sliding groove, and the sliding block is fixedly connected with the fixed rod and penetrates through the placing plate.

7. The method of claim 6, wherein the gene mutation is detected by the XNA molecular clamp-multiplex fluorescence PCR method,

the fixed rod comprises a sliding rod and a first spring, the sliding rod is connected with the placing plate in a sliding mode and located in the sliding groove, and the first spring is fixedly connected with the sliding rod, fixedly connected with the placing plate and located between the sliding rod and the placing plate.

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