CN108085391B - RNA quality control product of lung cancer fusion gene detection kit capable of being stably stored - Google Patents
RNA quality control product of lung cancer fusion gene detection kit capable of being stably stored Download PDFInfo
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Abstract
The invention discloses an RNA quality control product of a lung cancer fusion gene detection kit, which can be stably stored, and comprises the following components: a positive reference substance, a negative reference substance and a detection limit reference substance. The positive reference substance, the negative reference substance and the detection limit reference substance respectively contain corresponding fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the preservative is selected from glycine, epsilon-polylysine or cysteine. The RNA quality control product provided by the invention can effectively prevent the degradation of free RNA, provide a stable environment for RNA, prolong the storage time of RNA, and has the advantages of low preparation cost, stable performance, easy storage and the like, can effectively monitor the factors such as personnel operation, instrument state, kit effectiveness and the like, and greatly improves the accuracy and reliability of the detection kit.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to an RNA quality control product of a lung cancer fusion gene detection kit capable of being stably stored.
Background
Lung cancer is the most common malignant tumor with the highest morbidity and mortality worldwide, and seriously harms human health. The global epidemiological data of tumors in 2012 show that about 1410 ten thousand new cases of cancer and about 820 ten thousand deaths occur each year in the world, wherein the incidence of lung cancer is 182.5 ten thousand, and about 159.0 ten thousand deaths occur, which is the first case. Lung cancer is classified into non-small cell lung cancer (NSCLC), which accounts for about 80% of all lung cancers, and small cell lung cancer. Chromosomal and gene rearrangements are common genetic alterations in malignancies, and the first finding by Soda et al in 2007 of a fusion gene of an echinoderm microtubule-associated protein-like 4(EML4) gene with Anaplastic Lymphoma Kinase (ALK) in lung cancer patients confirms the presence of gene fusion in NSCLC. The ALK fusion gene is used as a new oncogene to participate in the development process of the NSCLC, the incidence rate of the ALK fusion gene is about 5 percent, and targeted inhibitors aiming at the ALK fusion gene, namely gefitinib, erlotinib and crizotinib, are widely applied to clinical treatment of the NSCLC at present. ROS1, RET fusion gene are also found in NSCLC.
Fusion gene screening has become an important and necessary indicator before targeted inhibitors are used to treat lung cancer. The national comprehensive cancer network clinical practice guideline, the lung cancer consensus of European medical oncology institute and the consensus of Chinese late non-small cell lung cancer molecular targeted therapy experts are all clearly suggested: before receiving treatment, NSCLC patients should detect EGFR, ALK and ROS1 genes, determine corresponding treatment strategies according to the gene states, and recommend that the detection of the three genes be carried out simultaneously under the condition permission.
Currently, methods for detecting lung cancer-driven fusion genes mainly include Fluorescence In Situ Hybridization (FISH), Immunohistochemistry (IHC), and real-time reverse transcription polymerase chain reaction (RT-PCR). A plurality of lung cancer fusion gene detection kits are developed based on the method, but at present, no economical, effective, stable and universal quality control product of the lung cancer fusion gene detection kit is sold in the market, the existing RNA quality control products mostly have the defects of instability, easy degradation, difficult storage and the like, and the quality control product is an important method and quality index for measuring and evaluating the detection accuracy, stability and other performances of the kit.
Disclosure of Invention
Based on the above, one of the purposes of the present invention is to overcome the defects of the prior art and provide an RNA quality control product of a lung cancer fusion gene detection kit, which has the advantages of stable storage, low manufacturing cost, stable performance, easy storage, effective monitoring of the factors such as personnel operation, instrument state, kit effectiveness, and the like, and greatly improve the accuracy and reliability of lung cancer fusion gene detection.
In order to achieve the purpose, the invention adopts the technical scheme that: an RNA quality control product of a lung cancer fusion gene detection kit capable of being stably stored, which comprises the following components: a positive reference, a negative reference and a detection limit reference;
the positive reference comprises the following components: the lung cancer fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride;
the negative reference comprises the following components: non-lung cancer fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride;
the detection limit reference substance comprises the following components: the lung cancer fusion gene RNA, the non-lung cancer fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride, wherein the mass ratio of the lung cancer fusion gene RNA to the total RNA is 1-5%;
the preservative is selected from glycine, epsilon-polylysine or cysteine.
Preferably, the molar concentrations of aurintricarboxylic acid ammonium salt, glycine and bestatin hydrochloride in the positive reference substance, the negative reference substance and the detection limit reference substance are as follows: 100-500 mu M of aurin-trimethyl ammonium salt, 80-180 mM of glycine and 20-60 nM of bestatin hydrochloride.
Preferably, the concentration of ammonium auridate in the positive reference, the negative reference and the detection limit reference is 200-400 mu M, the concentration of glycine is 90-110 mM, and the concentration of bestatin hydrochloride is 30-50 nM; more preferably, the concentration of aurin tricarbamate in the positive reference, the negative reference and the detection limit reference is 300 μ M, the concentration of glycine is 100mM, and the concentration of bestatin hydrochloride is 40 nM.
Preferably, the concentration of ammonium auridate in the positive reference substance, the negative reference substance and the detection limit reference substance is 100-500 mu M, the concentration of epsilon-polylysine is 2-4 mg/ml, and the concentration of bestatin hydrochloride is 20-60 nM.
Preferably, the aurintricarboxylic acid ammonium salt in the positive reference product, the negative reference product and the detection limit reference product is 200-400 mu M, the concentration of epsilon-polylysine is 2.5-3.5 mg/ml, and the phenylbutazone hydrochloride is 30-50 nM; more preferably, the concentration of the aurintricarboxylic acid ammonium salt in the positive reference substance, the negative reference substance and the detection limit reference substance is 300 mu M, the concentration of epsilon-polylysine is 3mg/ml and the concentration of the bestatin hydrochloride is 40 nM.
Preferably, the concentration of ammonium auricolinate in the positive reference, the negative reference and the detection limit reference is 100-500 mu M, the concentration of cysteine is 5-10 mM, and the concentration of bestatin hydrochloride is 20-60 nM.
Preferably, the aurintricarboxylic acid ammonium salt in the positive reference product, the negative reference product and the detection limit reference product is 200-400 mu M, the concentration of cysteine is 5-10 mM, and the concentration of bestatin hydrochloride is 30-50 nM; more preferably, the concentration of the aurintricarboxylic acid ammonium salt in the positive reference substance, the negative reference substance and the detection limit reference substance is 300 μ M, the concentration of cysteine is 7.5mM, and the concentration of bestatin hydrochloride is 40 nM.
Preferably, the positive reference is selected from at least one of an ALK fusion gene reference, a ROS1 fusion gene reference, and a RET fusion gene reference;
the ALK fusion gene reference comprises the following components: the ALK fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the ROS1 fusion gene reference comprises the following components: the ROS1 fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the RET fusion gene reference comprises the following components: the RET fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride.
Preferably, the negative reference is selected from at least one of a BCR-ABL fusion gene reference, an AML1-ETO fusion gene reference, and a CBF β -MYH11 fusion gene reference;
the BCR-ABL fusion gene reference comprises the following components: BCR-ABL fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the AML1-ETO fusion gene reference product comprises the following components: AML1-ETO fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the CBF beta-MYH 11 fusion gene reference product comprises the following components: AML1-ETO fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride.
Preferably, the ALK fusion gene reference in the positive reference is selected from at least one of ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6 and ALK-CLTC fusion gene references;
the ROS1 fusion gene reference is selected from at least one of ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN, and ROS1-TMEM106B fusion gene reference;
the RET fusion gene reference is selected from KIF5B-RET and RET-CCDC6 fusion gene reference.
Preferably, the ALK fusion gene reference in the positive reference is selected from the group consisting of ALK-EML4, ALK-KIF5B, and ALK-TFG;
the ROS1 fusion gene reference is selected from the group consisting of: ROS1-SLC34A2, ROS1-CD74, ROS1-EZR, and ROS1-SDC 4;
the RET fusion gene reference is selected from: RET-CCDC 6.
The positive reference substance is obtained by analyzing and screening a large number of samples in work of the inventor, and the fusion gene of the type is used as the positive reference substance, so that the lung cancer type of most Chinese people can be covered, a better reference effect is achieved, and the cost can be reduced.
The present invention also aims at providing the preparation method of the RNA quality control product of the lung cancer fusion gene detection kit capable of being stably preserved.
In order to achieve the above object, the present invention adopts a technical scheme that a method for preparing an RNA quality control product of a lung cancer fusion gene detection kit, which can be stably stored, is characterized by comprising the following steps:
(1) preparation of a positive reference: firstly, obtaining the lung cancer fusion gene through PCR amplification; carrying out enzyme digestion and connection on the lung cancer fusion gene and the vector, transforming competent cells, screening to obtain a positive bacterial colony containing the lung cancer fusion gene, and carrying out sequencing verification; thirdly, carrying out ultrasonic induction on the bacterial colony with the correct sequencing result to prepare a pseudovirus solution; fourthly, extracting RNA of the pseudovirus solution, and adding ammonium aurintricarboxylate, a preservative and phenylbutazone hydrochloride to obtain the pseudovirus solution;
(2) preparation of negative reference: firstly, obtaining a non-lung cancer fusion gene through PCR amplification; carrying out enzyme digestion and connection on the non-lung cancer fusion gene and the vector, transforming competent cells, screening to obtain a positive bacterial colony containing the non-lung cancer fusion gene, and carrying out sequencing verification; carrying out ultrasonic induction on the bacterial colony with the correct sequencing result to prepare a pseudovirus solution; fourthly, extracting RNA of the pseudovirus solution, and adding ammonium aurintricarboxylate, a preservative and phenylbutazone hydrochloride to obtain the pseudovirus solution;
(3) preparation of a detection limit reference substance: preparing the lung cancer fusion gene RNA according to the method in the step (1); preparing non-lung cancer fusion gene RNA according to the method in the step (2); mixing the lung cancer fusion gene RNA and the non-lung cancer fusion gene RNA to enable the mass ratio of the lung cancer fusion gene RNA to the total RNA to be 1-5%, and adding auric trimethyl ammonium salt, a preservative and phenylbutazone hydrochloride to obtain the lung cancer fusion gene RNA.
The invention also provides application of the RNA quality control product of the lung cancer fusion gene detection kit capable of being stably stored in preparation of the lung cancer fusion gene detection kit.
The inventor of the application finds that the addition of aurotriacetic acid ammonium salt, glycine and bestatin hydrochloride in the RNA quality control product can effectively protect the RNA in the quality control product, prevent the degradation of the RNA and prolong the quality guarantee period of the quality control product.
Compared with the prior art, the invention has the beneficial effects that: (1) the RNA quality control product provided by the invention contains RNA protection components, and each component and the using amount are obtained by a large amount of research and analysis of the inventor, so that the RNA degradation can be effectively prevented, the RNA quality control product is more stable and easier to store, and the effective period of the product is prolonged; (2) the RNA quality control product of the lung cancer fusion gene detection kit provided by the invention can effectively monitor the accuracy and stability of the kit, solve the problem of quality and performance evaluation of the kit, ensure the accuracy and reliability of screening of the lung cancer fusion gene, and simultaneously has the advantages of simplicity, convenience, low use cost and the like; (3) the RNA quality control product of the lung cancer fusion gene detection kit provided by the invention can monitor factors such as personnel operation, instrument state and the like on the premise of ensuring the effectiveness of the detection kit, and further ensures the accuracy of a detection result.
Drawings
FIG. 1 is a graph showing the size detection of RNA fragments of ALK-EML4 fusion gene RNA reference samples of Experimental group 3 after being stored at-80 ℃ for 2 weeks.
FIG. 2 is a graph showing the size detection of RNA fragments of ALK-EML4 fusion gene RNA reference of Experimental group 3 after being stored at 37 ℃ for 2 weeks.
FIG. 3 is a graph showing the size detection of RNA fragments of ALK-EML4 fusion gene RNA reference in control group 1 after being stored at-80 ℃ for 2 weeks.
FIG. 4 is a graph showing the size detection of RNA fragments of ALK-EML4 fusion gene RNA reference of control group 2 after being stored at 37 ℃ for 2 weeks.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
One embodiment of the RNA quality control product of the lung cancer fusion gene detection kit comprises a positive reference product, a negative reference product and a detection limit reference product.
The positive reference comprises an ALK fusion gene reference, an ROS1 fusion gene reference and an RET fusion gene reference. The ALK fusion gene reference comprises an ALK-EML4, an ALK-KIF5B, an ALK-KLC1, an ALK-TFG, an ALK-TPR, an ALK-HIP1, an ALK-STRN, an ALK-DCTN1, an ALK-SQSTM1, an ALK-BIRC6 and an ALK-CLTC fusion gene reference; the ROS1 fusion gene reference comprises ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN and ROS1-TMEM106B fusion gene reference; the RET fusion gene reference comprises KIF5B-RET and RET-CCDC6 fusion gene reference. The fusion gene reference product respectively contains corresponding fusion gene RNA, ammonium aurintricetate 100 mu M, glycine 80mM and bestatin hydrochloride 20 nM.
The negative reference comprises a BCR-ABL fusion gene reference, an AML1-ETO fusion gene reference and a CBF beta-MYH 11 fusion gene reference. The fusion gene reference product respectively contains corresponding fusion gene RNA, ammonium aurintricetate concentration of 100 mu M, epsilon-polylysine concentration of 2mg/ml and phenylbutazone hydrochloride concentration of 20 nM.
The detection limit reference comprises ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6, ALK-CLTC, ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS 3-EZR, ROS 3-LRIG 3, ROS 3-KDELR 3, ROS 3-LIMA 3, ROS 3-MSN, ROS 3-TMEM 106 3, KIF5 3-RET and RET-CCDC 3 fusion gene detection limit reference; the fusion gene detection limit reference substance comprises corresponding fusion gene RNA, BCR-ABL fusion gene RNA, AML1-ETO fusion gene RNA, CBF beta-MYH 11 fusion gene RNA, aurotriacetic acid ammonium salt with the concentration of 100 mu M, cysteine with the concentration of 5mM and phenylbutazone hydrochloride with the concentration of 20nM respectively, and the fusion gene RNA accounts for 2% of the total RNA by mass.
Example 2
One embodiment of the RNA quality control product of the lung cancer fusion gene detection kit comprises a positive reference product, a negative reference product and a detection limit reference product.
The positive reference comprises an ALK fusion gene reference, an ROS1 fusion gene reference and an RET fusion gene reference. The ALK fusion gene reference comprises an ALK-EML4, an ALK-KIF5B, an ALK-KLC1 and an ALK-BIRC6 fusion gene reference; the ROS1 fusion gene reference comprises ROS1-MSN, ROS1-EZR and ROS1-TMEM106B fusion gene reference; the RET fusion gene reference comprises a KIF5B-RET fusion gene reference. The fusion gene reference product respectively contains corresponding fusion gene RNA, aurin trimethyl ammonium salt 500 mu M, glycine 180mM and bestatin hydrochloride 60 nM.
The negative reference comprises an AML1-ETO fusion gene reference and a CBF beta-MYH 11 fusion gene reference. The fusion gene reference product respectively contains corresponding fusion gene RNA, aurin trimethyl ammonium salt with the concentration of 500 mu M, epsilon-polylysine with the concentration of 4mg/ml and phenylbutazone hydrochloride with the concentration of 60 nM.
The detection limit reference substance comprises an ALK-EML4, an ALK-KIF5B, an ALK-KLC1, an ALK-BIRC6, an ROS1-MSN, an ROS1-EZR, an ROS1-TMEM106B and a KIF5B-RET fusion gene detection limit reference substance; the fusion gene detection limit reference substance comprises corresponding fusion gene RNA, AML1-ETO fusion gene RNA, CBF beta-MYH 11 fusion gene RNA, aurin-trimethyl ammonium salt with the concentration of 500 mu M, cysteine with the concentration of 10mM and phenylbutazone hydrochloride with the concentration of 60nM respectively, and the fusion gene RNA accounts for 1% of the total RNA by mass.
Example 3
One embodiment of the RNA quality control product of the lung cancer fusion gene detection kit comprises a positive reference product, a negative reference product and a detection limit reference product.
The positive reference comprises an ALK fusion gene reference and an ROS1 fusion gene reference. The ALK fusion gene reference comprises an ALK-EML4, an ALK-KIF5B, an ALK-KLC1, an ALK-TFG, an ALK-TPR, an ALK-HIP1, an ALK-STRN, an ALK-DCTN1, an ALK-SQSTM1, an ALK-BIRC6 and an ALK-CLTC fusion gene reference; the ROS1 fusion gene reference comprises ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN and ROS1-TMEM106B fusion gene reference; the fusion gene reference product respectively contains corresponding fusion gene RNA, aurin trimethyl ammonium salt 400 mu M, glycine 110mM and bestatin hydrochloride 35 nM.
The negative reference comprises a BCR-ABL fusion gene reference, an AML1-ETO fusion gene reference and a CBF beta-MYH 11 fusion gene reference. The fusion gene reference product respectively contains corresponding fusion gene RNA, ammonium aurintricetate concentration of 400 mu M, epsilon-polylysine concentration of 2.5mg/ml and phenylbutazone hydrochloride concentration of 35 nM.
The detection limit reference comprises ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6, ALK-CLTC, ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN, and ROS1-TMEM106B detection limit reference; the fusion gene detection limit reference substance comprises corresponding fusion gene RNA, BCR-ABL fusion gene RNA, AML1-ETO fusion gene RNA, CBF beta-MYH 11 fusion gene RNA, aurotriacetic acid ammonium salt with the concentration of 400 mu M, cysteine with the concentration of 8mM and phenylbutazone hydrochloride with the concentration of 35nM respectively, and the fusion gene RNA accounts for 5% of the total RNA by mass.
Example 4
One embodiment of the RNA quality control product of the lung cancer fusion gene detection kit comprises a positive reference product, a negative reference product and a detection limit reference product.
The positive reference comprises an ALK fusion gene reference. The ALK fusion gene reference comprises an ALK-EML4, an ALK-KIF5B, an ALK-KLC1, an ALK-TFG, an ALK-TPR, an ALK-HIP1, an ALK-STRN, an ALK-DCTN1, an ALK-SQSTM1, an ALK-BIRC6 and an ALK-CLTC fusion gene reference. The fusion gene reference product contains corresponding fusion gene RNA, 300 mu M aurintricarboxylic acid ammonium salt, 100mM glycine and 40nM phenylbutazone hydrochloride.
The negative reference comprises a BCR-ABL fusion gene reference, an AML1-ETO fusion gene reference and a CBF beta-MYH 11 fusion gene reference. The fusion gene reference product contains corresponding fusion gene RNA, 300 mu M of aurin trimethyl ammonium salt, 3mg/ml of epsilon-polylysine and 40nM of phenylbutazone hydrochloride.
The detection limit reference comprises ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6 and ALK-CLTC detection limit reference; the fusion gene detection limit reference substance comprises corresponding fusion gene RNA, CBFbeta-MYH 11 fusion gene RNA, 300 mu M of aurin-trimethyl ammonium salt concentration, 7.5mM of cysteine concentration and 40nM of phenylbutazone hydrochloride concentration, and the mass ratio of the fusion gene RNA to the total RNA is 3%.
Example 5
The embodiment of the preparation method of the RNA quality control product of the lung cancer fusion gene detection kit comprises the following steps:
(1) preparation of a positive reference: preparing a target lung cancer fusion gene: and designing a sequence of a covering fracture point according to the target lung cancer fusion gene mRNA sequence in the NCBI database, and synthesizing the target lung cancer fusion gene. PCR amplification was performed using DNA containing the target lung cancer fusion gene sequence as a template, and the primer sequences are shown in Table 1. And (3) carrying out agarose gel electrophoresis on the PCR product, cutting gel on the band containing the target lung cancer fusion gene fragment, and recovering the target lung cancer fusion gene by using an agarose gel recovery kit. Secondly, constructing and verifying a plasmid vector containing the target lung cancer fusion gene: and (3) carrying out double enzyme digestion on the target lung cancer fusion gene fragment and the vector by using restriction endonucleases, carrying out agarose gel electrophoresis detection on the enzyme digestion product, and cutting and recovering the gel. And (3) connecting the enzyme digestion products to obtain a connecting product, then converting the connecting product into competent cells, coating the competent cells on an LB (Langmuir-Blodgett) culture medium plate, carrying out inverted culture at 37 ℃, and selecting a single white positive colony. Extracting DNA of the positive colony, carrying out PCR verification, and carrying out sequencing verification on the colony verified to be positive of the target lung cancer fusion gene. Preparing a target lung cancer fusion gene pseudovirus solution: and (3) inoculating the single positive colony confirmed by sequencing into an LB liquid culture medium, culturing, centrifuging, discarding the supernatant, and leaving a precipitate. Adding ultrasonic buffer (50mmol/L Tris-HCl, pH 8.0; 2mmol/L EDTA; 20% volume fraction glycerol; 100mmol/L NaCl; 0.1% volume fraction Triton X-100), and performing ice-bath ultrasonic treatment under the following ultrasonic conditions: 350W, intermittent 5s, ultrasonic 5s and 30 cycles. Then the ultrasonic product is centrifuged at 6000rpm for 10 minutes, and the supernatant is collected, namely the pseudovirus solution. Preparing quality control products: DNaseI was added to the pseudovirus solution and the DNA in the solution was digested. Extracting RNA of the pseudovirus solution by using a Trizol extraction method, and then adding ammonium aurotriacetate, glycine and bestatin hydrochloric acid to obtain the pseudovirus RNA.
(2) Preparation of negative reference: preparing a non-target lung cancer fusion gene: and designing a sequence of a covering fracture point according to the mRNA sequence of the non-target lung cancer fusion gene in the NCBI database, and synthesizing the non-target lung cancer fusion gene. PCR amplification was performed using DNA containing a non-target lung cancer fusion gene sequence as a template, and the primer sequences are shown in Table 1. And (3) carrying out agarose gel electrophoresis on the PCR product, cutting gel on the band containing the non-target lung cancer fusion gene fragment, and recovering the non-target lung cancer fusion gene by using an agarose gel recovery kit. Secondly, constructing and verifying a plasmid vector containing the non-target lung cancer fusion gene: and (3) carrying out double enzyme digestion on the non-target lung cancer fusion gene fragment and the vector by using restriction endonucleases, carrying out agarose gel electrophoresis detection on the enzyme digestion product, and cutting the gel for recovery. And (3) connecting the enzyme digestion products to obtain a connecting product, then converting the connecting product into competent cells, coating the competent cells on an LB (Langmuir-Blodgett) culture medium plate, carrying out inverted culture at 37 ℃, and selecting a single white positive colony. Extracting DNA of the positive colony, carrying out PCR verification, and carrying out sequencing verification on the colony verified to be positive of the non-target lung cancer fusion gene by the PCR. Preparing a non-target lung cancer fusion gene pseudovirus solution: and (3) inoculating the single positive colony confirmed by sequencing into an LB liquid culture medium, culturing, centrifuging, discarding the supernatant, and leaving a precipitate. Adding ultrasonic buffer (50mmol/L Tris-HCl, pH 8.0; 2mmol/L EDTA; 20% volume fraction glycerol; 100mmol/L NaCl; 0.1% volume fraction Triton X-100), and performing ice-bath ultrasonic treatment under the following ultrasonic conditions: 350W, intermittent 5s, ultrasonic 5s and 30 cycles. Then the ultrasonic product is centrifuged at 6000rpm for 10 minutes, and the supernatant is collected, namely the pseudovirus solution. Preparing quality control products: DNaseI was added to the pseudovirus solution and the DNA in the solution was digested. Extracting RNA of the pseudovirus solution by using a Trizol extraction method, and then adding ammonium aurometalate, epsilon-polylysine and bestatin hydrochloric acid to obtain the pseudovirus RNA.
(3) Preparation of a detection limit reference substance: preparing target lung cancer fusion gene RNA according to the method in the step (1); preparing non-target lung cancer fusion gene RNA according to the method in the step (2); mixing the target lung cancer fusion gene RNA and the non-target lung cancer fusion gene RNA to ensure that the mass ratio of the target lung cancer fusion gene RNA to the total RNA is 2 percent, and adding aurin-trimethyl ammonium salt, cysteine and bestatin hydrochloride to obtain the medicine.
TABLE 1 primer sequences
Example 6
In this example, the effect of preserving RNA in the RNA quality control of the present invention was investigated.
1. Design of experiments
Taking ALK-EML4 fusion gene reference in the positive reference as an example, the storage effect of RNA in the quality control product of the invention under different storage conditions is studied. Setting experimental groups 1-6 and control groups 1-2, wherein ALK-EML4 fusion gene reference products in the experimental groups 1-6 respectively contain ALK-EML4 fusion gene RNA, auric trimethyl ammonium salt, preservative (glycine, epsilon-polylysine and cysteine) and phenylbutazone hydrochloride, and ALK-EML4 fusion gene reference products in the control groups 1-2 respectively contain ALK-EML4 fusion gene RNA and DEPC water, and are specifically shown in Table 1:
TABLE 1 design of the experiment
2. Experimental methods
Pseudovirus solutions containing ALK-EML4 fusion genes were prepared as described in example 5, RNA in the pseudovirus solutions was extracted and equally divided into 8 groups, and an ALK-EML4 fusion gene reference sample was prepared for each group by adding aurintricarboxylic acid ammonium salt, glycine, and bestatin hydrochloride or DEPC water to the RNA solution according to the design shown in Table 1. RNA concentrations were determined and recorded for each group of ALK-EML4 fusion gene reference prior to initiation of the experiment. Then, storing each group of ALK-EML4 fusion gene reference products under corresponding conditions, and respectively measuring the size and the concentration of the RNA fragment of each group of ALK-EML4 fusion gene reference products by an Agilent 2100 bioanalyzer after 2 weeks so as to detect the stability and the degradation condition of each group of ALK-EML4 fusion gene reference products. Wherein, the detection of 3100-3200bp RNA fragment indicates that the ALK-EML4 fusion gene reference substance is not completely degraded, and the degradation rate of the ALK-EML4 fusion gene reference substance is calculated by the following formula: percent degradation ═ RNA concentration before experiment-RNA concentration after experiment)/RNA concentration before experiment × 100%.
3. Results of the experiment
After 2 weeks, the ALK-EML4 fusion gene reference products of each group were tested, and the RNA fragment size test results of experiment group 3, experiment group 6, control group 1 and control group 2 are shown in FIGS. 1, 2, 3 and 4, respectively, and the specific test results are shown in Table 2:
TABLE 2 results of the experiment
According to the experimental results, the ALK-EML4 fusion gene reference product provided by the invention comprises auricome tricarbamate, a preservative and bestatin hydrochloride, can obviously improve the stability of RNA at-80 ℃ (experimental groups 1-3) and 37 ℃ (experimental groups 4-6), and can effectively prevent the degradation of RNA in the preservation process, wherein the ALK-EML4 fusion gene reference products in the experimental group 3 and the experimental group 6 have the best preservation effect on RNA. Under the same preservation condition, the degradation rate of RNA in the reference products of the control groups 1-2 is obviously higher than that of the ALK-EML4 fusion gene reference product. Particularly, under the condition of being stored at 37 ℃, the RNA solution in the control group 2 is completely degraded after 2 weeks (the degradation rate is 100%), while the RNA degradation rate in the ALK-EML4 fusion gene reference product is below 30% (experiment groups 4-6). RNA in the ALK-EML4 fusion gene reference product in the embodiment 3 of the invention has no degradation condition within 2 weeks, and the effective period is 3 years and 6 months according to the basic principle and related method for determining the effective period of a medical instrument accelerated aging experiment. The preservation effect of RNA in other types of positive reference products, negative reference products and detection limit reference products is similar to that of the invention, and specific data are omitted.
Example 7
In this example, the RNA quality control product of the lung cancer fusion gene detection kit in example 1 is taken as an example to study the accuracy of the RNA quality control product.
1. Detection method
And analyzing the RNA quality control product by using a high-throughput target sequencing method, and evaluating the accuracy of the RNA quality control product.
2. The result of the detection
The results are shown in table 3:
TABLE 3 RNA quality control accuracy test results
According to the experimental results, the fusion genes in the positive reference substance, the negative reference substance and the detection limit reference substance can be accurately detected, and the types of the fusion genes completely accord with each other. The accuracy of the reference substance provided by the invention is 100%. The results of the detection of the RNA quality control products in examples 2 to 4 were the same as those in example 1, and the accuracy was 100%, and detailed data were omitted.
Example 8
In this example, the stability of the RNA quality control product of the lung cancer fusion gene detection kit in example 1 is taken as an example to study the stability of the RNA quality control product.
1. Detection method
And analyzing the randomly extracted three sets of RNA quality control products of different production batches by using a high-throughput targeted sequencing method.
2. The result of the detection
The results are shown in table 4:
TABLE 4 stability test results of RNA quality control products of three different batches
From the above results, the detection results of the three sets of RNA quality control products randomly extracted are accurate and the fusion genotype types are completely consistent, which indicates that the detection results of the three sets of RNA quality control products are not different and are completely correct. As the three sets of RNA quality control products are randomly extracted, the detection results of other RNA quality control products are also consistent, which shows that the RNA quality control product provided by the invention has stable performance. And the precision of the three sets of repetitive reference products meets the requirement that the coefficient of variation (CV,%) is not more than 15.0. The results of the detection of the RNA quality control products in examples 2 to 4 were the same as in example 1, and detailed data were omitted.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (7)
1. The RNA quality control product of the lung cancer fusion gene detection kit capable of being stably stored is characterized by comprising the following components: a positive reference, a negative reference and a detection limit reference;
the positive reference comprises the following components: the lung cancer fusion gene RNA, aurin trimethyl ammonium salt, preservative glycine and phenylbutazone hydrochloride;
the negative reference comprises the following components: non-lung cancer fusion gene RNA, aurin trimethyl ammonium salt, preservative epsilon-polylysine and phenylbutazone hydrochloride;
the detection limit reference substance comprises the following components: the lung cancer fusion gene RNA, the non-lung cancer fusion gene RNA, aurin trimethyl ammonium salt, preservative cysteine and phenylbutazone hydrochloride, wherein the mass ratio of the lung cancer fusion gene RNA to the total RNA is 1-5%;
in the positive reference substance, the molar concentrations of aurintricarboxylic acid ammonium salt, glycine and bestatin hydrochloride are respectively as follows: 100-500 mu M of aurin trimethyl ammonium salt, 80-180 mM of glycine and 20-60 nM of bestatin hydrochloride;
in the negative reference product, the concentration of aurintricarboxylic acid ammonium salt is 100-500 mu M, the concentration of epsilon-polylysine is 2-4 mg/ml, and the concentration of phenylbutazone hydrochloride is 20-60 nM;
the concentration of the ammonium aurintricetate salt in the detection limit reference product is 100-500 mu M, the concentration of the cysteine is 5-10 mM, and the concentration of the bestatin hydrochloride is 20-60 nM.
2. The RNA quality control of a stably preservable lung cancer fusion gene assay kit according to claim 1, wherein the positive reference is selected from at least one of an ALK fusion gene reference, an ROS1 fusion gene reference, and an RET fusion gene reference;
the ALK fusion gene reference comprises the following components: the ALK fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the ROS1 fusion gene reference comprises the following components: the ROS1 fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the RET fusion gene reference comprises the following components: the RET fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride.
3. The RNA quality control of a stably preservable lung cancer fusion gene detection kit according to claim 1, wherein the negative reference is selected from at least one of a BCR-ABL fusion gene reference, an AML1-ETO fusion gene reference, and a CBF β -MYH11 fusion gene reference;
the BCR-ABL fusion gene reference comprises the following components: BCR-ABL fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the AML1-ETO fusion gene reference product comprises the following components: AML1-ETO fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride; the CBF beta-MYH 11 fusion gene reference product comprises the following components: AML1-ETO fusion gene RNA, aurin trimethyl ammonium salt, preservative and phenylbutazone hydrochloride.
4. The RNA quality control of a stably preservable lung cancer fusion gene detection kit according to claim 2, wherein the ALK fusion gene reference in the positive reference is selected from at least one of ALK-EML4, ALK-KIF5B, ALK-KLC1, ALK-TFG, ALK-TPR, ALK-HIP1, ALK-STRN, ALK-DCTN1, ALK-SQSTM1, ALK-BIRC6, and ALK-CLTC fusion gene references;
the ROS1 fusion gene reference is selected from at least one of ROS1-CD74, ROS1-SLC34A2, ROS1-GOPC, ROS1-SDC4, ROS1-TPM3, ROS1-EZR, ROS1-LRIG3, ROS1-KDELR2, ROS1-LIMA1, ROS1-MSN, and ROS1-TMEM106B fusion gene reference;
the RET fusion gene reference is selected from KIF5B-RET and RET-CCDC6 fusion gene reference.
5. The RNA quality control of a stably preservable lung cancer fusion gene detection kit according to claim 4, wherein the ALK fusion gene reference in the positive reference is selected from the group consisting of ALK-EML4, ALK-KIF5B, and ALK-TFG;
the ROS1 fusion gene reference is selected from the group consisting of: ROS1-SLC34A2, ROS1-CD74, ROS1-EZR, and ROS1-SDC 4;
the RET fusion gene reference is selected from: RET-CCDC 6.
6. The method for preparing RNA quality control of a lung cancer fusion gene detection kit capable of being stably stored according to claim 1, comprising the steps of:
(1) preparation of a positive reference: firstly, obtaining the lung cancer fusion gene through PCR amplification; carrying out enzyme digestion and connection on the lung cancer fusion gene and the vector, transforming competent cells, screening to obtain a positive bacterial colony containing the lung cancer fusion gene, and carrying out sequencing verification; thirdly, carrying out ultrasonic induction on the bacterial colony with the correct sequencing result to prepare a pseudovirus solution; fourthly, extracting RNA of the pseudovirus solution, and adding ammonium aurintricarboxylate, a preservative and phenylbutazone hydrochloride to obtain the pseudovirus solution;
(2) preparation of negative reference: firstly, obtaining a non-lung cancer fusion gene through PCR amplification; carrying out enzyme digestion and connection on the non-lung cancer fusion gene and the vector, transforming competent cells, screening to obtain a positive bacterial colony containing the non-lung cancer fusion gene, and carrying out sequencing verification; carrying out ultrasonic induction on the bacterial colony with the correct sequencing result to prepare a pseudovirus solution; fourthly, extracting RNA of the pseudovirus solution, and adding ammonium aurintricarboxylate, a preservative and phenylbutazone hydrochloride to obtain the pseudovirus solution;
(3) preparation of a detection limit reference substance: preparing the lung cancer fusion gene RNA according to the method in the step (1); preparing non-lung cancer fusion gene RNA according to the method in the step (2); mixing the lung cancer fusion gene RNA and the non-lung cancer fusion gene RNA to enable the mass ratio of the lung cancer fusion gene RNA to the total RNA to be 1-5%, and adding auric trimethyl ammonium salt, a preservative and phenylbutazone hydrochloride to obtain the lung cancer fusion gene RNA.
7. Use of the RNA quality control of the lung cancer fusion gene detection kit capable of being stably stored according to claim 1 in preparation of the lung cancer fusion gene detection kit.
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