Device capable of rapidly detecting malignant hyperpyrexia and application thereof

Abstract

The invention provides a device capable of rapidly detecting malignant hyperpyrexia and application of the device. Specifically, the invention provides a method for detecting the malignant hyperpyrexia related gene locus, which comprises the following steps: (1) amplifying the malignant hyperpyrexia related gene locus in a sample by adopting a specific primer to obtain an amplified product; (2) carrying out library establishment and sequencing on the amplified product to obtain sequencing data of the malignant hyperpyrexia related gene locus; the working concentration of each specific primer ranges from 0.005 [mu] M to 0.05 [mu] M; the amplification condition is as follows: the temperature increasing and decreasing rate of PCR annealing and extension is 1 DEG C / sec to 4 DEG C / sec.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a device capable of rapidly detecting malignant hyperthermia and an application thereof. Background Art

[0002] Malignant hyperthermia (MH) is a familial (autosomal dominant) clinical syndrome with typical clinical manifestations of abnormal skeletal muscle hypermetabolism triggered mainly by volatile inhalation anesthetics and succinylcholine. Potent volatile anesthetics are widely used and generally safe induction agents for general anesthesia. Agents include sevoflurane, halothane, enflurane, isoflurane, methoxyflurane, and desflurane, but all currently available potent inhalation anesthetics may cause malignant hyperthermia.

[0003] Malignant hyperthermia is the only known hereditary disease that can cause perioperative death due to conventional anesthetic drugs. MH-susceptible individuals have abnormal skeletal muscle cells and calcium ion regulation disorders. Under the action of triggering factors (mainly volatile inhaled anesthetics and succinylcholine), calcium ion release increases abnormally and cannot be effectively reabsorbed, resulting in abnormal increase in intracellular calcium ion levels and a series of hypermetabolic syndromes. In the absence of specific therapeutic drugs, general clinical cooling measures are difficult to control the increase in body temperature, which can eventually lead to the death of patients. The incidence of MH attacks is estimated to be between 1 / 10,000 and 1 / 250,000. It is estimated that the prevalence of MH genetic traits is between 1 / 2,000 and 1 / 3,000.

[0004] The etiology of MH is very complex. Current studies have shown that physiologically, it is caused by abnormal ion channel defects that lead to the release of a large amount of Ca2+ from the sarcoplasmic reticulum into the cytoplasm, triggering sustained muscle contraction, accompanied by a significant increase in heat production, leading to a series of clinical manifestations. From the molecular level of gene analysis, RYR1 is the most important. The RYR1 gene is located at 19q13.1-19q13.2 and consists of 160,000 base pairs encoding 160 exons. Its encoded protein is Ca 2+ Due to the defective function of RYR1 receptor in MH patients, the channel is continuously opened under the triggering of certain anesthetic drugs, and a large amount of Ca 2+ outflow from the sarcoplasmic reticulum, far exceeding the Ca 2+ The recovery capacity of the pump causes the muscles to continue to contract strongly.

[0005] Another CACNAIS gene is located at lq32, with 93,500 base pairs encoding 44 exons, which determine the amino acid sequence of the α1 subunit of the dihydropyridine Ca2+ channel (dihydropyridine receptor, DHPR). DHPR is located on the transverse tubule membrane of muscle cells and is a voltage-gated channel. When the sarcolemma depolarizes, the transmembrane potential is rapidly transmitted to the transverse tubule membrane, exciting DHPR and ultimately causing the RYR1 receptor to open, Ca 2+ Flowing out from the sarcoplasmic reticulum. Therefore, the three are closely related in space and function.

[0006] The current gold standard for diagnosing MH is the caffeine-halothane isolated skeletal muscle contraction test, which is relatively invasive and requires professional personnel to operate. This test can only be performed in research laboratories and has not yet been popularized in clinical practice. Experts from all regions agree that the key to perioperative management of susceptible patients or suspected susceptible patients is prevention first to avoid MH attacks.

[0007] Currently, the detection methods for malignant hyperthermia include gene chips, first-generation sequencing and other methods, but the high cost of gene chip detection and low first-generation sequencing throughput limit the large-scale promotion of malignant hyperthermia detection. Summary of the invention

[0008] In order to solve the deficiencies in the prior art, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for detecting malignant hyperthermia-related gene loci, comprising:

[0010] (1) using specific primers to amplify the malignant hyperthermia-related gene loci in the sample to obtain the amplified products;

[0011] (2) constructing a library and sequencing the amplified products to obtain sequencing data of malignant hyperthermia-related gene loci;

[0012] The working concentration of each specific primer is 0.005 μM-0.05 μM;

[0013] The amplification conditions include:

[0014] The heating and cooling rates for PCR annealing and extension are 1°C / sec-4°C / sec.

[0015] According to a specific embodiment of the present invention, the sequencing adopts the second generation sequencing technology.

[0016] According to a specific embodiment of the present invention, the working concentration of each specific primer is 0.01 μM-0.04 μM. As a preferred embodiment, the concentration of each specific primer is 0.02 μM.

[0017] According to a specific embodiment of the present invention, the heating and cooling rates of PCR annealing and extension are 1.5°C / sec-3.5°C / sec.

[0018] As a preferred embodiment, the heating and cooling rates of the PCR annealing and extension are: heating rate 3°C / sec, cooling rate 2°C / sec.

[0019] According to a specific embodiment of the present invention, the sample includes an oral mucosa sample and a blood sample.

[0020] According to a specific embodiment of the present invention, the malignant hyperthermia-related gene loci include the RYR1 gene loci and / or the CACNAIS gene loci. As a preferred embodiment, the malignant hyperthermia-related gene loci include chr1:201061121, chr1:201029943, chr19:38924507, chr19:38931442, chr19:38931469, chr19:38933074, chr19:38934851, chr19:38934852, chr19:38934893, chr19:38937350, chr19:38939313, chr 19:38939352、chr19:38942482、chr19:38942483、chr19:38942490、chr19:38945999、chr19:38946103、chr19:38946111、chr19:38946112、chr19: 38946129, chr19:38946168, chr19:38948179, chr19:38948185, chr19:38 948186, chr19:38976478, chr19:38985204, chr19:38985205, chr19:3898 5219, chr19:38986918, chr19:38986923, chr19:38986934, chr19:38989863, chr19:38990289-38990291, chr19:38990295, chr19:38990310, chr1 9:38990337, chr19:38990371, chr19:38990615, chr19:38990624, chr19: 38990633, chr19:38990637, chr19:38990643, chr19:38991276, chr19:38 991282, chr19:38991283, chr19:38991294, chr19:38991295, chr19:38991538, chr19:38991539, chr19:38994959, chr19:39002961, chr19:39025 415, chr19:39034005, chr19:39034472, chr19:39052003, chr19:3905567 4. chr19:39068594, chr19:39068595, chr19:39070644, chr19:39070728,chr19:39070734, chr19:39070754, chr19:39071010, chr19:39071043, chr19:39071080, chr19:39071125, chr19:39075629 and / or chr19:39076780.

[0021] In a specific embodiment of the present invention, the chr19:38937350 has two variations: NM_000540.3:c.742G>C and NM_000540.3:c.742G>A.

[0022] In a specific embodiment of the present invention, the chr19:38939352 has two variations: NM_000540.3:c.1021G>C and NM_000540.3:c.1021G>A.

[0023] In a specific embodiment of the present invention, the chr19:38945999 has two variations: NM_000540.3:c.1565A>C and NM_000540.3:c.1565A>G.

[0024] In a specific embodiment of the present invention, the chr19:38985205 has two variations: NM_000540.3:c.6488G>A and NM_000540.3:c.6488G>C.

[0025] In a specific embodiment of the present invention, the chr19:38986923 has two variations: NM_000540.3:c.6617C>G and NM_000540.3:c.6617C>T.

[0026] In a specific embodiment of the present invention, the chr19:38990637 has two variations: NM_000540.3:c.7304G>A and NM_000540.3:c.7304G>T.

[0027] In a specific embodiment of the present invention, the chr19:39055674 has two variations: NM_000540.3:c.12700G>C and NM_000540.3:c.12700G>T.

[0028] According to a specific embodiment of the present invention, the sequence of the specific primer is shown as SEQ ID NO.1-84.

[0029] In a second aspect, the present invention provides a reagent composition for detecting malignant hyperthermia-related gene loci, comprising:

[0030] Reagent materials used to amplify malignant hyperthermia-related gene loci in samples;

[0031] Reagents and materials used for next-generation sequencing.

[0032] In a specific embodiment of the present invention, the reagent material for amplifying the malignant hyperthermia-related gene loci in the sample is a primer that specifically targets the malignant hyperthermia-related gene loci.

[0033] In a specific embodiment of the present invention, the reagent materials used for the second-generation sequencing are the reagent materials in the Yikon kit (Gene Sequencing Universal Library Kit XK-008-24).

[0034] In a third aspect, the present invention provides a device for detecting malignant hyperthermia, comprising:

[0035] an amplification module, used to process the sample to be tested according to the method described in the first aspect of the present invention to obtain sequencing data of malignant hyperthermia-related gene loci;

[0036] The data analysis module analyzes and processes the sequencing data.

[0037] As a preferred embodiment, the device further comprises a result output module for outputting the malignant hyperthermia analysis result.

[0038] In a fourth aspect, the present invention provides a computer storage medium storing computer program instructions, which, when executed, implement: detecting malignant hyperthermia-related gene loci using the method described in the first aspect of the present invention.

[0039] In a fifth aspect, the present invention provides a computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the computer program, the method described in the first aspect of the present invention is used to detect gene loci associated with malignant hyperthermia.

[0040] In a sixth aspect, the present invention provides the use of a reagent for detecting malignant hyperthermia-related gene loci in the preparation of a device for detecting malignant hyperthermia. The use includes the process of detecting malignant hyperthermia-related gene loci using the method described in the first aspect of the present invention.

[0041] Beneficial effects:

[0042] The present invention performs targeted amplification on 73 RYR1 gene loci and 2 CACNAIS gene loci, and relies on a second-generation sequencing platform to detect malignant hyperthermia at the gene molecular level, thereby providing a detection method to reduce the occurrence of malignant hyperthermia in potential high-risk patients.

[0043] In the present invention, oral mucosal samples can be directly amplified without the need for a nucleic acid extraction process, which can greatly save experimental operation time. Targeted amplification experiments can be performed directly on oral mucosal samples, and sequencing can be performed directly on the machine after the amplification is completed. Based on the analysis results of the sites, it can be determined whether the target sites in the sample to be tested have mutated. The purpose is to help clinical anesthesiologists provide effective information.

[0044] The present invention is simple and fast, and 75 sites can be carried out simultaneously in the same PCR tube. Compared with the traditional multiplex PCR library construction process, multiple purification operations in the experimental process are reduced. The traditional multiplex PCR detection process includes: specific primer amplification → product purification → non-specific product enzymatic hydrolysis → product purification → PCR product enrichment → product purification. The present invention adjusts the optimal working concentration of the specific primers of the sites to be tested, and combines the optimal heating and cooling speed during the PCR amplification process, and can directly adjust the experimental process to: specific primer amplification → PCR product enrichment → product purification. It can directly skip the non-specific product enzymatic hydrolysis process and multiple purification operations in the traditional multiplex PCR process, and the operability is greatly improved and the entire experimental process can be effectively shortened. The detection cost is low and the sample collection is convenient, which is convenient for large-scale promotion among the population. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Show the amplification principle involved in the present invention;

[0046] Figure 2A The results were verified for the JYD generation of samples;

[0047] Figure 2B The results are verified for the LJ generation of samples;

[0048] Figure 2C The results were verified for the HHH generation of samples;

[0049] Figure 2D Verification results for the sample HRX generation. DETAILED DESCRIPTION

[0050] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0051] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0052] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field.

[0053] The invention involves information on 75 sites, as shown in Table 1, and the site amplification primer sequences are shown in Table 2.

[0054] Table 1

[0055]

[0056]

[0057]

[0058]

[0059] “ / ” in the table means there is no relevant value.

[0060] Table 2

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] The working concentration of each primer in the present invention is shown in Table 3.

[0069] Table 3

[0070]

[0071] Wherein, concentration B is the preferred working concentration of each primer of the present invention, concentration A is the working concentration of each primer in the low-concentration primer combination, and concentration C is the working concentration of each primer in the high-concentration primer combination. The working concentration of each primer in Example 2 and Example 3 is concentration B.

[0072] During the amplification process, the heating and cooling rates of PCR annealing and extension are shown in Table 4.

[0073] Table 4

[0074]

[0075] Among them, VB is the preferred heating and cooling rate of the present invention, rate VA is a low rate combination, and rate VC is a high rate combination. The heating and cooling rates in Examples 2 and 3 are VB, the heating rate is 3°C / sec, and the cooling rate is 2°C / sec.

[0076] The sample detection method is shown in Table 5. The reagents used in the present invention are all components of the Yikang kit (Gene Sequencing Universal Library Kit XK-008-24).

[0077] Table 5

[0078]

[0079]

[0080] Example 1

[0081] The three groups of primers with concentrations A, B and C were optimized and compared in combination with the heating and cooling rates VA, VB and VC during the PCR process, so as to obtain the most suitable rapid detection method for the present invention. DNA samples extracted from human peripheral blood were amplified, with 2 replicates in each group, and the amplified products were sequenced on the second generation machine. The specific experimental scheme is shown in Table 6.

[0082] Table 6

[0083]

[0084] Detailed sequencing quality control data are shown in Table 7.

[0085] Table 7

[0086]

[0087]

[0088] Example 2

[0089] Oral mucosa samples: Use commercially available oral swab collection kits to collect samples, and the collection method follows the operating instructions. Centrifuge the collected oral mucosa samples at 2000×g for 10 min at room temperature, discard the supernatant completely, resuspend the cell pellet in 1 ml of nuclease-free water or PBS buffer, centrifuge at 2000×g for 10 min at room temperature, discard the supernatant completely, repeat the cell resuspending once, and wash for the last time to remove as much residual liquid as possible. See Table 5 for the specific experimental process.

[0090] Results Analysis

[0091] The specific detection results of the samples are shown in Table 8.

[0092] Table 8

[0093]

[0094]

[0095]

[0096]

[0097] During the testing of sample ZJF, no mutations were found at any sites, suggesting that the sample was not susceptible to malignant hyperthermia.

[0098] Example 3

[0099] The method of the present invention was used to detect 24 peripheral blood samples from clinical patients suspected of malignant anesthesia hyperthermia. After peripheral blood nucleic acid extraction was completed for the 24 samples, the specific primer amplification stage was directly entered. The specific experimental process is shown in Table 5.

[0100] Results Analysis

[0101] Among the 24 peripheral blood samples, 4 samples had mutation sites detected, and 19 samples had no mutation sites detected. The specific detection information of the samples is shown in Table 9. Sample JYD mutated at site RYR1, c.7523G>A, sample LJ mutated at RYR1, c.7361G>A, sample HHH mutated at RYR1, c.1021G>A, and sample HRX mutated at RYR1, c.1021G>A. The mutation sites of the above samples were amplified by first-generation sequencing specific primers (see Table 10 for site primer sequences). After verification by first-generation sequencing, the results were consistent with the detection results of the present invention (see Figure 2A-2D ), indicating that the sample may be a population susceptible to malignant hyperthermia. In this example, all sites in Table 1 were tested using first-generation sequencing, and mutations that were not detected using the method of the present invention were not detected by first-generation sequencing.

[0102] Table 9

[0103]

[0104]

[0105] Table 10

[0106]

[0107] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for detecting malignant hyperthermia-related gene loci, comprising: (1) using specific primers to amplify the malignant hyperthermia-related gene loci in the sample to obtain the amplified products; (2) constructing a library and sequencing the amplified products to obtain sequencing data of malignant hyperthermia-related gene loci; The working concentration of each specific primer is 0.005 μM-0.05 μM; The amplification conditions include: The heating and cooling rates for PCR annealing and extension are 1°C / sec-4°C / sec.

2. The method according to claim 1, wherein: The sequencing adopts the second generation sequencing technology.

3. The method according to claim 1, wherein: The working concentration of each specific primer is 0.01 μM-0.04 μM.

4. The method according to claim 1, wherein: The heating and cooling rates of the PCR annealing and extension are 1.5°C / sec-3.5°C / sec.

5. The method according to claim 1, wherein: The samples include oral mucosa samples and blood samples.

6. The method according to any one of claims 1 to 5, wherein: The malignant hyperthermia-related gene loci include the RYR1 gene loci and / or the CACNAIS gene loci. Preferably, the malignant hyperthermia-related gene loci include chr1:201061121, chr1:201029943, chr19:38924507, chr19:38931442, chr19:38931469, chr19:38933074, chr19:38934851, chr19:38934852, chr19:38934893, chr19:38937350, chr19:38939313, chr19:38939352, chr19:38942 482、chr19:38942483、chr19:38942490、chr19:38945999、chr19:3894610 3. chr19:38946111, chr19:38946112, chr19:38946129, chr19:38946168, chr19:38948179、chr19:38948185、chr19:38948186、chr19:38976478、chr19:38985204、chr19:38985205、chr19:38985219、chr19:38986918、chr19 :38986923、chr19:38986934、chr19:38989863、chr19:38990289-3899029 1. chr19:38990295, chr19:38990310, chr19:38990337, chr19:38990371, chr19:38990615、chr19:38990624、chr19:38990633、chr19:38990637、chr19:38990643、chr19:38991276、chr19:38991282、chr19:38991283、chr19 :38991294、chr19:38991295、chr19:38991538、chr19:38991539、chr19:3 8994959, chr19:39002961, chr19:39025415, chr19:39034005, chr19:3903 4472, chr19:39052003, chr19:39055674, chr19:39068594, chr19:390685 95. chr19:39070644, chr19:39070728, chr19:39070734, chr19:39070754,chr19:39071010, chr19:39071043, chr19:39071080, chr19:39071125, chr19:39075629 and / or chr19:39076780.

7. A reagent composition for detecting malignant hyperthermia-related gene loci, comprising: Reagent materials used to amplify malignant hyperthermia-related gene loci in samples; Reagents and materials used for next-generation sequencing.

8. A device for detecting malignant hyperthermia, comprising: an amplification module, used to process the sample to be tested according to the method according to any one of claims 1 to 6 to obtain sequencing data of malignant hyperthermia-related gene loci; Data analysis module, which analyzes and processes sequencing data; Preferably, the device further comprises a result output module for outputting the malignant hyperthermia analysis result.

9. A computer storage medium storing computer program instructions, wherein when the computer program instructions are executed, the method according to any one of claims 1 to 6 is used to detect gene loci associated with malignant hyperthermia.

10. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein: When the processor executes the computer program, it realizes: detecting malignant hyperthermia-related gene loci using the method described in any one of claims 1 to 6.

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