WO2022120917A1 - Cox6c检测引物及其应用 - Google Patents

Cox6c检测引物及其应用 Download PDF

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WO2022120917A1
WO2022120917A1 PCT/CN2020/137129 CN2020137129W WO2022120917A1 WO 2022120917 A1 WO2022120917 A1 WO 2022120917A1 CN 2020137129 W CN2020137129 W CN 2020137129W WO 2022120917 A1 WO2022120917 A1 WO 2022120917A1
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cox6c
detection
incubate
reaction
optionally
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陈宇
屈雪琪
林力
原琳
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深圳先进技术研究院
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present application belongs to the technical field of gene detection, and in particular relates to Cox6c detection primers and applications thereof.
  • AD Alzheimer's disease
  • the quality of life of the patients has dropped sharply and they are unable to take care of themselves.
  • there is no specific drug or cure for the disease and the long treatment and care of the patients has brought a huge burden to the patients' families and the society.
  • AD Alzheimer's disease
  • diagnosis methods of AD are mainly combined diagnosis, which mainly include: neuropsychological assessment, cognitive impairment test, brain senile plaque and Tau protein PET scan and brain magnetic resonance (MRI) and cerebrospinal fluid (CSF) markers, beta - Amyloid and phosphorylated tau protein detection, etc.
  • MRI brain magnetic resonance
  • CSF cerebrospinal fluid
  • AD Alzheimer's disease
  • early molecular screening techniques for AD include positron emission tomography (PET) and cerebrospinal fluid A ⁇ molecular level detection. cause surgical infection.
  • PET positron emission tomography
  • cerebrospinal fluid A ⁇ molecular level detection cause surgical infection.
  • the accuracy of these diagnostic techniques for the early diagnosis of AD is also unstable, and it is difficult to be used for early AD screening.
  • CN105969885A discloses a genetic diagnosis kit for Alzheimer's disease, by designing and synthesizing 10 pathogenic genes of Alzheimer's disease (APP, APOE, PSEN1, PSEN2, GSK3 ⁇ , DYRK1A, Tomm40, CLU, PICALM and TAU) specific primers, by collecting the specimens of the individual to be tested, using RT-PCR technology, amplifying each gene to obtain specific DNA products for sequencing analysis, and then comparing the sample sequence with the normal gene sequence to analyze whether there is any consistency disease mutations to assess individual risk.
  • This method is simple and fast, has good specificity and high sensitivity, but requires auxiliary nucleic acid sequencing technology and has high cost, which is not suitable for practical clinical screening.
  • the diagnosis technology for Alzheimer's disease often has the problems of difficult operation and poor accuracy of early screening. How to provide an early diagnosis technology of Alzheimer's disease, which can quickly diagnose or diagnose the disease in the early stage of the patient's disease. It has become an urgent problem to enable potential patients to detect risks in advance and take targeted treatment or prevention.
  • the present application provides Cox6c detection primers and applications.
  • the Cox6c detection primers have good specificity and high sensitivity; the Cox6c detection kit is easy to use, simple to operate, short detection time, high accuracy, and has extremely high application value .
  • the application provides Cox6c detection primers, and the Cox6c detection primers include the nucleic acid sequences shown in SEQ ID No. 1-2;
  • SEQ ID No. 1 GGGAAGGACGTTGGTGTAGA;
  • SEQ ID No. 2 CCAGCAATATGAACCCGCAG.
  • Cox6c is the gene encoding cytochrome C oxidase subunit 6c.
  • Cytochrome C oxidase is located at the end of the mitochondrial respiratory chain and is an important component of mitochondrial oxidation reaction and release of energy for the body to use.
  • Cox6c is directly related to the signaling pathways of electron transport, ATP generation and heat release in respiration.
  • the Cox6c detection primers described in this application are of suitable length and good specificity, and there is no mismatch in the amplification process; the amplification products are of reasonable size and can be used as detection primers to detect the expression level of Cox6c; the detection reaction has high sensitivity and accuracy Good performance and high application value.
  • the present application provides a Cox6c detection kit, the Cox6c detection kit includes the Cox6c detection primer described in the first aspect.
  • the Cox6c detection kit includes the Cox6c detection primer, which can be used to detect the expression level of Cox6c; the kit contains the necessary reagents in the detection experiment, which is convenient to use and simple to operate; the Cox6c described in this application
  • the detection kit can be used for both qualitative detection and quantitative detection.
  • the detection object can be cells, tissues, etc. There are many types of samples that can be detected, and the application range is wide.
  • the Cox6c detection kit further includes a reverse transcription reaction reagent and a detection reaction reagent.
  • the reverse transcription reaction reagents include reverse transcription primers, reverse transcriptase, dNTPs, RNase inhibitors and reverse transcription buffer.
  • the reverse transcription primers include Oligo dT and/or random primers.
  • the detection reaction reagent includes DNA polymerase, fluorescent dye and PCR buffer.
  • the fluorescent dye includes SYBR.
  • the Cox6c detection kit further includes deionized water, and the deionized water includes deionized water without RNase pollution.
  • the Cox6c detection kit further includes an internal reference gene primer.
  • the internal reference gene primers include GAPDH amplification primers and/or HPRT amplification primers.
  • the amplification primers for GAPDH include the nucleic acid sequences shown in SEQ ID No. 3-4.
  • SEQ ID No. 3 TCAACAGCAACTCCCACTCTTCCA;
  • SEQ ID No. 4 ACCCTGTTGCTGTAGCCGTATTCA.
  • the amplification primers for HPRT include the nucleic acid sequences shown in SEQ ID No. 5-6.
  • SEQ ID No. 6 GGGACGCAGCAACTGACATTTCTA.
  • the present application provides a method for detecting the expression level of Cox6c, the detection method comprising using the Cox6c detection primers described in the first aspect and/or using the Cox6c detection kit described in the second aspect, detecting in the sample
  • the expression levels of Cox6c were analyzed statistically.
  • the Cox6c detection primer has high sensitivity and good specificity; the Cox6c detection kit can detect many types of samples, and can perform quantitative detection. By collecting samples of different cells and different tissues at different time points , using the reverse transcription reaction reagent and detection reaction reagent in the kit to detect the expression of Cox6c in the sample, and at the same time with the help of the internal reference gene primer in the kit, the relative expression level of Cox6c in the sample can be obtained. After statistical analysis, Corresponding conclusions can be drawn, the operation is simple and the accuracy is high.
  • the expression level of Cox6c in the detection sample includes reverse transcription reaction and detection reaction.
  • the reverse transcription reaction comprises:
  • reaction mixture I The template, reverse transcription primer and water are mixed, and ice bath is carried out after incubation to obtain reaction mixture I;
  • Reverse transcriptase, dNTPs, RNase inhibitor and reverse transcription buffer were added to the reaction mixture I, and a reverse transcription reaction was performed to obtain the first strand cDNA.
  • the template comprises an RNA template.
  • the mixing further includes a centrifugation step, and the centrifugation time is 3-5s, for example, 3s, 3.5s, 4s, 4.5s or 5s.
  • the incubation temperature is 65°C to 75°C, such as 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C, Preferably it is 70 degreeC.
  • the incubation time is 3-7 min, for example, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min or 7 min, preferably 5 min.
  • the time of the ice bath is not less than 2min, for example, it can be 2min, 2.5min, 3min, 3.5min, 4min, 4.5min or 5min.
  • the program of the reverse transcription reaction is:
  • Incubate at 23 to 28°C for 4 to 6 minutes can be, for example, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C or 28°C.
  • the time may for example be 4min, 4.5min, 5min, 5.5min or 6min;
  • Incubate at 40 to 45°C for 55 to 65 minutes can be, for example, 40°C, 40.5°C, 41°C, 41.5°C, 42°C, 42.5°C, 43°C, 43.5°C, 44°C, 44.5°C or 45°C.
  • the time can be, for example, 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62min, 63min, 64min or 65min;
  • Incubate at 68 to 72°C for 3 to 7 minutes can be, for example, 68°C, 68.5°C, 69°C, 69.5°C, 70°C, 70.5°C, 71°C, 71.5°C or 72°C, and the incubation time can be, for example, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min or 7min.
  • the detection reaction includes:
  • the first-strand cDNA, Cox6c detection primer, internal reference gene primer, DNA polymerase, fluorescent dye, PCR buffer and water were mixed, and after centrifugation, the detection reaction was performed.
  • the rotational speed of the centrifugation is 5500-6500 rpm, such as 5500 rpm, 5600 rpm, 5700 rpm, 5800 rpm, 5900 rpm, 6000 rpm, 6100 rpm, 6200 rpm, 6300 rpm, 6400 rpm or 6500 rpm, preferably 6000 rpm.
  • the centrifugation time is 55-65s, for example, 55s, 56s, 57s, 58s, 59s, 60s, 61s, 62s, 63s, 64s or 65s.
  • the procedure of described detection reaction is:
  • Pre-denaturation incubate at 94-98°C for 8-12 minutes, the incubation temperature can be, for example, 94°C, 94.5°C, 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C or 98°C, and the incubation time can be, for example, is 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min or 12min;
  • the incubation temperature can be, for example, 94°C, 94.5°C, 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C or 98°C
  • the incubation time can be, for example, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s or 30s;
  • Incubate at 55 to 65°C for 30 to 60s can be, for example, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C or 65°C.
  • the time can be, for example, 30s, 35s, 40s, 45s, 50s, 55s or 60s;
  • the incubation temperature can be, for example, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C.
  • the time may for example be 0.5min, 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min or 5min;
  • the incubation temperature can be, for example, 93°C, 93.5°C, 94°C, 94.5°C, 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C or 98°C.
  • the time can be, for example, 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s or 30s;
  • the incubation temperature can be, for example, 58°C, 58.5°C, 59°C, 59.5°C, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C or 63°C.
  • the time may be, for example, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 65s, 70s, 75s, 80s, 85s or 90s.
  • the number of cycles of amplification is 30 to 45 times, such as 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, 38 times, 39 times, 40 times times, 41 times, 42 times, 43 times, 44 times or 45 times, preferably 40 times.
  • the rate of heating and cooling in the detection reaction process is 1.5-1.8°C/s, for example, 1.5°C/s, 1.6°C/s, 1.7°C/s or 1.8°C/s.
  • the step of diluting the first-strand cDNA is further included.
  • the step of obtaining the sample and extracting RNA is further included.
  • the sample includes neural synapses and/or peripheral blood mononuclear cells.
  • the method for detecting the expression level of Cox6c described in this application specifically includes the following steps:
  • reaction mixture I RNA template, Oligo dT and/or random primers with water, centrifuge for 3 to 5 s, incubate at 65 to 75°C for 3 to 7 min, and ice bath for at least 2 min to obtain reaction mixture I;
  • Dilute the first-strand cDNA Dilute the first-strand cDNA, mix the diluted first-strand cDNA, Cox6c detection primer, internal reference gene primer, DNA polymerase, fluorescent dye, PCR buffer and water, and centrifuge at 5500-6500 rpm for 55-65 s to perform the detection reaction ;
  • Pre-denaturation incubate at 94-98°C for 8-12min;
  • Cyclic amplification incubate at 94-98°C for 15-30s; incubate at 55-65°C for 30-60s; incubate at 65-75°C for 0.5-5min;
  • the number of times of the cyclic amplification is 30 to 45 times;
  • the rate of heating and cooling in the detection reaction process is 1.5-1.8°C/s;
  • the application provides a detection device for Cox6c expression level, the detection device includes an RNA extraction unit, a reverse transcription unit, a detection unit and an analysis unit;
  • the RNA extraction unit is used for extracting RNA from the collected neural synapses and/or peripheral blood mononuclear cell samples;
  • the reverse transcription unit is used for reverse transcription of the obtained RNA into first-strand cDNA
  • the detection unit is used to detect the obtained first-strand cDNA
  • the analysis unit is used to perform statistical analysis on the result of the detection reaction.
  • reaction mixture I Preferably, in the reverse transcription unit, the template, the reverse transcription primer and water are mixed, and an ice bath is performed after incubation to obtain reaction mixture I;
  • the reaction mixture I is further mixed with reverse transcriptase, dNTPs, RNase inhibitor and reverse transcription buffer to carry out reverse transcription reaction to obtain the first strand cDNA.
  • the template comprises an RNA template.
  • the mixing further includes a centrifugation step, and the centrifugation time is 3-5s, for example, 3s, 3.5s, 4s, 4.5s or 5s.
  • the incubation temperature is 65°C to 75°C, such as 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C, Preferably it is 70 degreeC.
  • the incubation time is 3-7 min, for example, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min or 7 min, preferably 5 min.
  • the time of the ice bath is not less than 2min, for example, it can be 2min, 2.5min, 3min, 3.5min, 4min, 4.5min or 5min.
  • the program of the reverse transcription reaction is:
  • Incubate at 23 to 28°C for 4 to 6 minutes can be, for example, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C or 28°C.
  • the time may for example be 4min, 4.5min, 5min, 5.5min or 6min;
  • Incubate at 40 to 45°C for 55 to 65 minutes can be, for example, 40°C, 40.5°C, 41°C, 41.5°C, 42°C, 42.5°C, 43°C, 43.5°C, 44°C, 44.5°C or 45°C.
  • the time can be, for example, 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62min, 63min, 64min or 65min;
  • Incubate at 68 to 72°C for 3 to 7 minutes can be, for example, 68°C, 68.5°C, 69°C, 69.5°C, 70°C, 70.5°C, 71°C, 71.5°C or 72°C, and the incubation time can be, for example, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min or 7min.
  • the first-strand cDNA, Cox6c detection primer, internal reference gene primer, DNA polymerase, fluorescent dye, PCR buffer and water are mixed, centrifuged, and the detection reaction is performed.
  • the rotational speed of the centrifugation is 5500-6500 rpm, such as 5500 rpm, 5600 rpm, 5700 rpm, 5800 rpm, 5900 rpm, 6000 rpm, 6100 rpm, 6200 rpm, 6300 rpm, 6400 rpm or 6500 rpm, preferably 6000 rpm.
  • the centrifugation time is 55-65s, for example, 55s, 56s, 57s, 58s, 59s, 60s, 61s, 62s, 63s, 64s or 65s.
  • the procedure of described detection reaction is:
  • Pre-denaturation incubate at 94-98°C for 8-12 minutes, the incubation temperature can be, for example, 94°C, 94.5°C, 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C or 98°C, and the incubation time can be, for example, is 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min or 12min;
  • the incubation temperature can be, for example, 94°C, 94.5°C, 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C or 98°C
  • the incubation time can be, for example, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s or 30s;
  • Incubate at 55 to 65°C for 30 to 60s can be, for example, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C or 65°C.
  • the time can be, for example, 30s, 35s, 40s, 45s, 50s, 55s or 60s;
  • the incubation temperature can be, for example, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C.
  • the time may for example be 0.5min, 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min or 5min;
  • the incubation temperature can be, for example, 93°C, 93.5°C, 94°C, 94.5°C, 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C or 98°C.
  • the time can be, for example, 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s or 30s;
  • the incubation temperature can be, for example, 58°C, 58.5°C, 59°C, 59.5°C, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C or 63°C.
  • the time may be, for example, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 65s, 70s, 75s, 80s, 85s or 90s.
  • the number of cycles of amplification is 30 to 45 times, such as 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, 38 times, 39 times, 40 times times, 41 times, 42 times, 43 times, 44 times or 45 times, preferably 40 times.
  • the rate of heating and cooling in the detection reaction process is 1.5-1.8°C/s, for example, 1.5°C/s, 1.6°C/s, 1.7°C/s or 1.8°C/s.
  • the step of diluting the first-strand cDNA is further included.
  • the application provides the Cox6c detection primer described in the first aspect, the Cox6c detection kit described in the second aspect, the detection method of the Cox6c expression level described in the third aspect, or the fourth aspect.
  • Cox6c is closely related to the body's respiration and energy metabolism, and neurodegenerative diseases represented by Alzheimer's disease are often accompanied by a state of low glucose metabolism, which has an important relationship with the mitochondrial respiratory chain. Therefore, Cox6c There is a certain correlation between the expression level of ⁇ - ⁇ and the pathogenesis of neurodegenerative diseases, and it can be used in the preparation of diagnostic reagents and the screening of related drugs.
  • the Cox6c detection primers described in this application have good specificity and high sensitivity, and are used in the related detection of Cox6c; the Cox6c detection kit containing the primers can be used to qualitatively or quantify various samples such as cells, tissues and organs
  • the detection method of Cox6c has a wide range of applications; the method for detecting the expression level of Cox6c is simple to operate and can be operated in most laboratories. High, high sensitivity, short time-consuming, low detection cost;
  • the expression level of Cox6c gene is related to energy metabolism in the body and the course of AD.
  • the cerebral cortex, hippocampus, brain synapse and peripheral blood of 3-month-old mice and 6-month-old mice
  • the expression level of Cox6c in mononuclear cells did not change significantly, while the expression level of Cox6c in different tissues of transgenic AD mice at different times changed significantly, and the expression level decreased in cerebral cortex, hippocampus and peripheral blood mononuclear cells.
  • RNA-seq and gene function enrichment show that Cox6c is related to neurodegenerative diseases, so it can be used in the preparation of diagnostic reagents for neurodegenerative diseases and related drug screening.
  • the method described in the present application has lower cost, shorter time and simpler operation, and has broad application prospects.
  • FIG. 1A is a volcano plot of gene differential distribution of 3-month-old wild-type mice and transgenic AD mice in Example 1 of the application;
  • Figure 1B is a volcano plot of the gene differential distribution of 6-month-old wild-type mice and transgenic AD mice in Example 1 of the application;
  • Figure 1C is a heat map of significantly different genes between wild-type mice and transgenic AD mice in Example 1 of the application;
  • 1D is a picture of the result of enrichment of significantly different genes in disease-related signaling pathways in Example 1 of the application;
  • Figure 2A shows the cerebral cortex (Cortex) and hippocampus (Hippocampus) of wild-type mice (WT) and transgenic AD mice (AD) at 3 months of age (3M) and 6 months of age (6M) in Example 4 of the present application Picture of the detection result of the relative expression level of Cox6c in ;
  • FIG. 2B shows Cox6c expression in brain synapses (SD) of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and transgenic AD mice (AD) in Example 4 of the present application The picture of the detection result of the relative expression level;
  • FIG 3 shows the cell stimulation of peripheral blood mononuclear cells (PBMCs) of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and transgenic AD mice (AD) in Example 5 of the present application Picture of the detection result of the relative expression level of Cox6c after stimulation with phorbol lipid (PMA).
  • PBMCs peripheral blood mononuclear cells
  • 6M 6-month-old wild-type mice
  • AD transgenic AD mice
  • Transgenic AD mice were purchased from Jackson Laboratory;
  • TRIzol was purchased from Invitrogen;
  • OptiPrep gradient centrifuge was purchased from Thermo Fisher Scientific;
  • the tissue homogenate balance solution is self-prepared, and the final concentration is 320mmol/L sucrose solution, 10mmol/L tris(hydroxymethyl)aminomethane hydrochloride solution, 1mmol/L ethylenediaminetetraacetic acid solution, 2.5mmol/L /L mixed solution of dithiothreitol solution and 0.25mmol/L sodium fluoride solution, wherein sucrose, tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, dithiothreitol and Sodium fluoride was purchased from Sigma-Aldrich;
  • Reverse transcription reaction reagents were purchased from Thermo Fisher Scientific;
  • Detection reagents were purchased from Thermo Fisher Scientific;
  • DPBS was purchased from Gibco;
  • RNA-seq service is provided by Nuohe Zhiyuan
  • RPMI-1640 was purchased from Gibco.
  • This example uses RNA-seq technology to detect differential expression in brain synapses (SD) of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and transgenic AD mice (AD) Gene analysis was carried out to compare the differences between wild-type and AD-type mice, to screen genes with a false discovery rate (FDR, false discovery rate) ⁇ 0.01, and to perform gene enrichment analysis for genes with a relative gene expression difference of more than 2 times. (GO analysis), the results are shown in Figure 1A, Figure 1B and Figure 1C.
  • Figure 1A shows the gene differences between 3-month-old wild-type mice and transgenic AD mice
  • Figure 1B shows the gene differences between 6-month-old wild-type mice and transgenic AD mice.
  • the heat map as shown in Fig. 1C, showed that there were significant differences in the expression levels of 12 genes, including Cox6c.
  • the expression of Cox6c in the brain nerve synapses of 3-month-old transgenic AD mice was lower than that of wild-type mice.
  • the expression of Cox6c was significantly increased in synapses.
  • This example provides Cox6c detection primers, the sequences of which are shown in SEQ ID No. 1-2:
  • SEQ ID No. 1 GGGAAGGACGTTGGTGTAGA;
  • SEQ ID No. 2 CCAGCAATATGAACCCGCAG.
  • the Cox6c detection primer has strong specificity and high sensitivity, and the size of the amplification product is 111 bp, which can be used in the qualitative or quantitative detection of Cox6c.
  • This embodiment provides a Cox6c detection kit, which includes a reverse transcription reagent and a detection reaction reagent.
  • the reverse transcription reagents include Oligo dT, random primers, reverse transcriptase, dNTPs, RNase inhibitors, reverse transcription buffer and deionized water without RNase pollution;
  • the detection reaction reagents include Cox6c detection primer SEQ ID No.1 ⁇ 2, SEQ ID No.3 ⁇ 4 of the amplification primer of the internal reference gene GAPDH, SEQ ID No.5 ⁇ 6 of the amplification primer of the internal reference gene HPRT, a mixture of DNA polymerase, SYBR and PCR buffer solution and RNase-free deionized water.
  • SEQ ID No. 3 TCAACAGCAACTCCCACTCTTCCA;
  • SEQ ID No. 4 ACCCTGTTGCTGTAGCCGTATTCA;
  • SEQ ID No. 6 GGGACGCAGCAACTGACATTTCTA.
  • the Cox6c detection kit described in this application contains the necessary reagents in the Cox6c detection experiment, which can be used for both qualitative detection and quantitative detection, and can detect various samples such as cells, tissues or organs, and has a wide range of applications. .
  • the Cox6c detection kit prepared in Example 3 was used to detect the cerebral cortex (Cortex) of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and transgenic AD mice (AD).
  • the expression levels of Cox6c in the hippocampus (Hippocampus) and brain synapses (SD) are as follows:
  • AD mice After 3-month-old and 6-month-old littermates of wild-type and transgenic AD mice were anesthetized with isoflurane gas, they were quickly decapitated with scissors after neck dislocation, and their heads were placed on ice, and the cerebral cortex and cerebral cortex of the mice were rapidly separated. hippocampus. The isolated tissue was washed twice in DPBS containing 4 U/mL of protease inhibitor and RNase inhibitor.
  • tissue homogenate balance solution containing protease inhibitors and RNase inhibitors
  • tissue homogenate balance solution containing protease inhibitors and RNase inhibitors
  • the filtered tissue homogenate was centrifuged at 10,000 ⁇ g for 10 min at 4°C to collect the precipitate;
  • the pellet was suspended in 35% OptiPrep tissue homogenate equilibration solution, and then placed in centrifuge tubes filled with 9%, 12.5%, 15%, 25% and 35% OptiPrep gradient centrifuges at 10,000°C at 4°C. Gradient centrifugation at ⁇ g for 24 min, carefully collect the suspension between the 9% and 12.5% interface, that is, the suspension containing brain synapses;
  • the collected brain synaptosome suspension was centrifuged at 6000 ⁇ g for 30 min at 4°C, and the obtained precipitate was the brain nerve synapse.
  • RNA in the aqueous phase Precipitate the RNA in the aqueous phase with isopropanol, add 0.5 mL of isopropanol for every 1 mL of TRIzol used, and place at room temperature for 10 min;
  • RNA precipitate with 75% ethanol solution, add 1 mL of 75% ethanol solution for every 1 mL of TRIzol used, centrifuge at 7500 ⁇ g for 5 min at 4°C, and remove the supernatant;
  • RNA precipitate was dried at room temperature for 5 min, and 25 ⁇ L of RNase-free deionized water was added to dissolve the RNA.
  • RNA obtained in step (1) was used as a template for reverse transcription reaction, and reaction mixture I was prepared in a 0.2 mL centrifuge tube without RNase contamination.
  • the components and additions are shown in Table 2.
  • reaction mixture I was mixed, centrifuged for 5 s, incubated at 70° C. for 5 min, ice-bathed for 5 min, and then added the reaction mixture II to the reaction mixture I.
  • the components and addition amounts of the reaction mixture II are shown in Table 3.
  • step (2) Using the first-strand cDNA obtained in step (2) as a template, the expression level of Cox6c in the corresponding sample is detected, and the specific steps are as follows:
  • the first-strand cDNA was diluted 10-fold, and the reaction system was configured, as shown in Table 4.
  • the reaction system was mixed, centrifuged at 6000rpm for 1min, and the detection reaction was carried out:
  • Pre-denaturation incubate at 95°C for 10min;
  • Cyclic amplification incubate at 95°C for 15s; incubate at 60°C for 60s; incubate at 70°C for 1 min;
  • Form a melting curve incubate at 95°C for 15s; incubate at 60°C for 60s;
  • the number of cycles is 40;
  • the rate of heating and cooling during the detection reaction was 1.6°C/s.
  • the disease course of the 3-month-old transgenic AD mice corresponds to the stage of mild intellectual impairment (MCI), while the 6-month-old transgenic AD mice corresponds to the stage of the development of clear Alzheimer's disease characteristics.
  • MCI mild intellectual impairment
  • 6-month-old transgenic AD mice corresponds to the stage of the development of clear Alzheimer's disease characteristics.
  • the Cox6c detection kit prepared in Example 3 was used to detect peripheral blood mononuclear cells of 3-month-old (3M) and 6-month-old (6M) wild-type mice (WT) and transgenic AD mice (AD) (PBMCs) the expression level of Cox6c after stimulation with the cell stimulator phorbol lipid (PMA), the specific steps are as follows:
  • the blood sample was carefully added to the liquid surface of the separation solution, and centrifuged at 400 ⁇ g for 20 min at 18 °C;
  • Peripheral blood mononuclear cells were seeded in a 6-well cell culture plate at a density of 1 ⁇ 10 6 /mL. After culturing for 24 hours, the cells were treated with 100 ⁇ PMA. Cells were harvested after stimulation for 24 h, and RNA extraction was performed.
  • the present application provides a detection primer for Cox6c, the primer has good specificity and high sensitivity; the Cox6c detection kit containing the primer can detect a variety of samples, and the operation is simple and the time is short. , a wide range of applications; the expression level of Cox6c in the body is closely related to the course of neurodegenerative diseases, therefore, the Cox6c detection primers and the Cox6c detection kit can be used in the preparation of diagnostic reagents for neurodegenerative diseases and corresponding drugs In the screening, the application value is extremely high.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

Cox6c检测引物及其应用，所述Cox6c检测引物包括SEQ ID No.1～2所示的核酸序列；还提供了一种Cox6c检测试剂盒，所述Cox6c检测试剂盒包括所述的Cox6c检测引物。所述Cox6c检测引物特异性好，灵敏度高；所述Cox6c检测试剂盒使用方便，可对多种样品进行定性或定量检测。使用所述的Cox6c检测引物和/或所述的Cox6c检测试剂盒，可以用来检测样本中的Cox6c的表达水平，操作简单，准确性高，在制备神经退行性疾病诊断试剂和/或筛选神经退行性疾病治疗药物中具有潜在的应用价值。

Description

Cox6c检测引物及其应用 技术领域
本申请属于基因检测技术领域，尤其涉及Cox6c检测引物及其应用。
背景技术
阿尔兹海默症(AD)是一种常见的老年慢性病，患者出现记忆损伤并最终引起痴呆。患者生活质量大幅下降并且出现生活不能自理的情况，现在还未有针对该病症的特效药或治愈手段，对患者的漫长的治疗和护理给患者家属和社会带来了巨大负担。
由于AD病程是一个不可逆的过程，因此，AD治疗的关键是早期诊断，在疾病早期对AD进行干预并延缓病程进展。但至今尚未有一种精准的方法可对AD进行早期预测或诊断。目前AD的诊断方法主要是联合诊断，主要包括：神经心理学评估、认知损伤测试、脑部老年斑块和Tau蛋白PET扫描和脑部核磁共振(MRI)和脑脊液(CSF)标志物、β-淀粉样蛋白和磷酸化tau蛋白检测等。由于AD早期症状不明显，通常对病症做出明确诊断时，患者多到达病程晚期，多数神经元已经死亡。如果能在病人发病早期进行快速诊断或使潜在病人提前发现风险，针对性地进行治疗或预防，有助于患者病程滞留在轻微智力损伤阶段(mild cognitive impairment，MCI)而减缓恶化，从而提高病人的生活质量并减轻社会负担。
对AD进行早期临床诊断和相应治疗是解决AD病症的关键点。现阶段对AD的早期分子筛查技术包括正电子发射型计算机断层显像(PET)和脑脊液Aβ分子水平检测等，前者要对受检者注射一定剂量的放射性物质，后者操作损伤大，易造成外科感染。此外，这些诊断技术针对AD早期诊断的准确性也不稳定，很难用于AD早期筛查。
一些检测方法及相应的试剂盒为阿尔兹海默症的诊断与鉴定提供了参考。CN105969885A公开了一种阿尔兹海默症的基因诊断试剂盒，通过设计并合成阿尔兹海默症的10个致病基因(APP、APOE、PSEN1、PSEN2、GSK3β、DYRK1A、Tomm40、CLU、PICALM和TAU)的特异性引物，通过采集待测个体的标本，运用RT-PCR技术，扩增各基因得到特异性DNA产物进行测序分析，然后将样 本序列与正常基因序列进行比对，分析是否有致病突变，以评估个体患病风险。该方法简便快捷，特异性好，灵敏度高，但需要辅助核酸测序技术，成本较高，不适合应用于实际的临床筛选中。
目前，针对阿尔兹海默症的诊断技术往往存在操作难度大及早期筛查准确性较差的问题，如何提供一种阿尔兹海默症的早期诊断技术，可以在病人发病早期进行快速诊断或使潜在病人提前发现风险，采取针对性的治疗或预防，已成为亟待解决的问题。
发明内容
本申请提供了Cox6c检测引物及应用，所述Cox6c检测引物的特异性好，灵敏度高；所述Cox6c检测试剂盒使用方便，操作简单，检测耗时短，准确度高，具有极高的应用价值。
为达此目的，本申请采用如下技术方案：
第一方面，本申请提供了Cox6c检测引物，所述Cox6c检测引物包括SEQ ID No.1～2所示的核酸序列；
SEQ ID No.1：GGGAAGGACGTTGGTGTAGA；
SEQ ID No.2：CCAGCAATATGAACCCGCAG。
本申请中，Cox6c是细胞色素C氧化酶亚基6c的编码基因。细胞色素C氧化酶位于线粒体的呼吸链末端，是线粒体进行氧化反应、释放能量供机体使用的重要组分，Cox6c与呼吸的电子转运、ATP生成及热量释放的信号通路有直接关联。本申请所述Cox6c检测引物的长度合适，特异性好，扩增过程中无错配现象；扩增产物的大小合理，可作为检测引物，用于检测Cox6c的表达水平；检测反应灵敏度高，准确性好，应用价值极高。
第二方面，本申请提供了一种Cox6c检测试剂盒，所述Cox6c检测试剂盒包括第一方面所述的Cox6c检测引物。
本申请中，所述Cox6c检测试剂盒包括所述的Cox6c检测引物，可以用来检测Cox6c的表达水平；试剂盒中包含检测实验中所必需的试剂，使用方便，操作简单；本申请所述Cox6c检测试剂盒既可以用于定性检测，又可以用于定量检测，检测对象可以为细胞、组织等，可检测的样本的类型较多，应用范围较广。
优选地，所述Cox6c检测试剂盒还包括反转录反应试剂和检测反应试剂。
优选地，所述反转录反应试剂包括反转录引物、反转录酶、dNTPs、RNA酶抑制剂和反转录缓冲液。
优选地，所述反转录引物包括Oligo dT和/或随机引物。
优选地，所述检测反应试剂包括DNA聚合酶、荧光染料和PCR缓冲液。
优选地，所述荧光染料包括SYBR。
优选地，所述Cox6c检测试剂盒还包括去离子水，所述去离子水包括无RNA酶污染的去离子水。
优选地，所述Cox6c检测试剂盒还包括内参基因引物。
优选地，所述内参基因引物包括GAPDH的扩增引物和/或HPRT的扩增引物。
优选地，所述GAPDH的扩增引物包括SEQ ID No.3～4所示的核酸序列。
SEQ ID No.3：TCAACAGCAACTCCCACTCTTCCA；
SEQ ID No.4：ACCCTGTTGCTGTAGCCGTATTCA。
优选地，所述HPRT的扩增引物包括SEQ ID No.5～6所示的核酸序列。
SEQ ID No.5：GGAGTCCTGTTGATGTTGCCAGTA；
SEQ ID No.6：GGGACGCAGCAACTGACATTTCTA。
第三方面，本申请提供了一种Cox6c表达水平的检测方法，所述检测方法包括使用第一方面所述的Cox6c检测引物和/或使用第二方面所述的Cox6c检测试剂盒，检测样本中的Cox6c的表达水平，并进行统计学分析。
本申请中，所述Cox6c的检测引物灵敏度高，特异性好；所述Cox6c检测试剂盒可以检测的样本类型较多，且可以进行定量检测，通过收集不同细胞、不同组织在不同时间点的样本，采用试剂盒中的反转录反应试剂和检测反应试剂对样本中Cox6c的表达量进行检测，同时借助于试剂盒中的内参基因引物，可获得样本中Cox6c的相对表达水平，统计分析后，即可得出相应结论，操作简便，准确度高。
优选地，所述检测样本中的Cox6c的表达水平包括反转录反应和检测反应。
优选地，所述反转录反应包括：
将模板、反转录引物和水混合，孵育后进行冰浴，得到反应混合物I；
向所述反应混合物I中加入反转录酶、dNTPs、RNA酶抑制剂和反转录缓 冲液，进行反转录反应，得到第一链cDNA。
优选地，所述模板包括RNA模板。
优选地，所述混合后还包括离心的步骤，所述离心的时间为3～5s，例如可以是3s、3.5s、4s、4.5s或5s。
优选地，所述孵育的温度为65～75℃，例如可以是65℃、66℃、67℃、68℃、69℃、70℃、71℃、72℃、73℃、74℃或75℃，优选为70℃。
优选地，所述孵育的时间为3～7min，例如可以是3min、3.5min、4min、4.5min、5min、5.5min、6min、6.5min或7min，优选为5min。
优选地，所述冰浴的时间为不少于2min，例如可以是2min、2.5min、3min、3.5min、4min、4.5min或5min。
优选地，所述反转录反应的程序为：
23～28℃孵育4～6min，孵育的温度例如可以是23℃、23.5℃、24℃、24.5℃、25℃、25.5℃、26℃、26.5℃、27℃、27.5℃或28℃，孵育的时间例如可以是4min、4.5min、5min、5.5min或6min；
40～45℃孵育55～65min，孵育的温度例如可以是40℃、40.5℃、41℃、41.5℃、42℃、42.5℃、43℃、43.5℃、44℃、44.5℃或45℃，孵育的时间例如可以是55min、56min、57min、58min、59min、60min、61min、62min、63min、64min或65min；
68～72℃孵育3～7min，孵育的温度例如可以是68℃、68.5℃、69℃、69.5℃、70℃、70.5℃、71℃、71.5℃或72℃，孵育的时间例如可以是3min、3.5min、4min、4.5min、5min、5.5min、6min、6.5min或7min。
优选地，所述检测反应包括：
将第一链cDNA、Cox6c检测引物、内参基因引物、DNA聚合酶、荧光染料、PCR缓冲液和水混合，离心后，进行检测反应。
优选地，所述离心的转速为5500～6500rpm，例如可以是5500rpm、5600rpm、5700rpm、5800rpm、5900rpm、6000rpm、6100rpm、6200rpm、6300rpm、6400rpm或6500rpm，优选为6000rpm。
优选地，所述离心的时间为55～65s，例如可以是55s、56s、57s、58s、59s、60s、61s、62s、63s、64s或65s。
优选地，所述检测反应的程序为：
预变性：94～98℃孵育8～12min，孵育的温度例如可以是94℃、94.5℃、95℃、95.5℃、96℃、96.5℃、97℃、97.5℃或98℃，孵育的时间例如可以是8min、8.5min、9min、9.5min、10min、10.5min、11min、11.5min或12min；
循环扩增：
94～98℃孵育15～30s，孵育的温度例如可以是94℃、94.5℃、95℃、95.5℃、96℃、96.5℃、97℃、97.5℃或98℃，孵育的时间例如可以是15s、16s、17s、18s、19s、20s、21s、22s、23s、24s、25s、26s、27s、28s、29s或30s；
55～65℃孵育30～60s，孵育的温度例如可以是55℃、56℃、57℃、58℃、59℃、60℃、61℃、62℃、63℃、64℃或65℃，孵育的时间例如可以是30s、35s、40s、45s、50s、55s或60s；
65～75℃孵育0.5～5min，孵育的温度例如可以是65℃、66℃、67℃、68℃、69℃、70℃、71℃、72℃、73℃、74℃或75℃，孵育的时间例如可以是0.5min、1min、1.5min、2min、2.5min、3min、3.5min、4min、4.5min或5min；
形成熔解曲线：
93～98℃孵育10～30s，孵育的温度例如可以是93℃、93.5℃、94℃、94.5℃、95℃、95.5℃、96℃、96.5℃、97℃、97.5℃或98℃，孵育的时间例如可以是10s、11s、12s、13s、14s、15s、16s、17s、18s、19s、20s、21s、22s、23s、24s、25s、26s、27s、28s、29s或30s；
58～63℃孵育30～90s，孵育的温度例如可以是58℃、58.5℃、59℃、59.5℃、60℃、60.5℃、61℃、61.5℃、62℃、62.5℃或63℃，孵育的时间例如可以是30s、35s、40s、45s、50s、55s、60s、65s、70s、75s、80s、85s或90s。
优选地，所述循环扩增的次数为30～45次，例如可以是30次、31次、32次、33次、34次、35次、36次、37次、38次、39次、40次、41次、42次、43次、44次或45次，优选为40次。
优选地，所述检测反应过程中升温和降温的速率为1.5～1.8℃/s，例如可以是1.5℃/s、1.6℃/s、1.7℃/s或1.8℃/s。
优选地，所述检测反应前还包括对第一链cDNA进行稀释的步骤。
优选地，在所述检测样本中的Cox6c的表达水平前还包括获取样本并提取RNA的步骤。
优选地，所述样本包括神经突触和/或外周血单个核细胞。
作为优选技术方案，本申请所述Cox6c表达水平的检测方法，具体包括以下步骤：
(1)收集神经突触和/或外周血单个核细胞样本，并提取RNA；
(2)反转录反应：
将RNA模板、Oligo dT和/或随机引物和水混合，离心3～5s，在65～75℃下孵育3～7min后，冰浴至少2min，得到反应混合物I；
向所述反应混合物I中加入反转录酶、dNTPs、RNA酶抑制剂和反转录缓冲液，进行反转录反应，程序为：
23～28℃孵育4～6min；
40～45℃孵育55～65min；
68～72℃孵育3～7min；
通过上述反应，得到第一链cDNA；
(3)检测反应：
对第一链cDNA进行稀释，将稀释后的第一链cDNA、Cox6c检测引物、内参基因引物、DNA聚合酶、荧光染料、PCR缓冲液和水混合，5500～6500rpm离心55～65s，进行检测反应；
所述检测反应的程序为：
预变性：94～98℃孵育8～12min；
循环扩增：94～98℃孵育15～30s；55～65℃孵育30～60s；65～75℃孵育0.5～5min；
形成熔解曲线：93～98℃孵育10～30s；58～63℃孵育30～90s；
所述循环扩增的次数为30～45次；
所述检测反应过程中升温和降温的速率为1.5～1.8℃/s；
(4)对检测反应的结果进行统计学分析。
第四方面，本申请提供了一种Cox6c表达水平的检测装置，所述检测装置包括RNA提取单元、反转录单元、检测单元和分析单元；
所述RNA提取单元用于从收集的神经突触和/或外周血单个核细胞样本中提取RNA；
所述反转录单元用于将所得RNA反转录成第一链cDNA；
所述检测单元用于对所得第一链cDNA进行检测；
所述分析单元用于对检测反应的结果进行统计学分析。
优选地，在所述反转录单元中，模板、反转录引物和水混合，孵育后进行冰浴，得到反应混合物I；
所述反应混合物I进一步与反转录酶、dNTPs、RNA酶抑制剂和反转录缓冲液混合，进行反转录反应，得到第一链cDNA。
优选地，所述模板包括RNA模板。
优选地，所述混合后还包括离心的步骤，所述离心的时间为3～5s，例如可以是3s、3.5s、4s、4.5s或5s。
优选地，所述孵育的温度为65～75℃，例如可以是65℃、66℃、67℃、68℃、69℃、70℃、71℃、72℃、73℃、74℃或75℃，优选为70℃。
优选地，所述孵育的时间为3～7min，例如可以是3min、3.5min、4min、4.5min、5min、5.5min、6min、6.5min或7min，优选为5min。
优选地，所述冰浴的时间为不少于2min，例如可以是2min、2.5min、3min、3.5min、4min、4.5min或5min。
优选地，所述反转录反应的程序为：
23～28℃孵育4～6min，孵育的温度例如可以是23℃、23.5℃、24℃、24.5℃、25℃、25.5℃、26℃、26.5℃、27℃、27.5℃或28℃，孵育的时间例如可以是4min、4.5min、5min、5.5min或6min；
40～45℃孵育55～65min，孵育的温度例如可以是40℃、40.5℃、41℃、41.5℃、42℃、42.5℃、43℃、43.5℃、44℃、44.5℃或45℃，孵育的时间例如可以是55min、56min、57min、58min、59min、60min、61min、62min、63min、64min或65min；
68～72℃孵育3～7min，孵育的温度例如可以是68℃、68.5℃、69℃、69.5℃、70℃、70.5℃、71℃、71.5℃或72℃，孵育的时间例如可以是3min、3.5min、4min、4.5min、5min、5.5min、6min、6.5min或7min。
优选地，在所述检测单元中，第一链cDNA、Cox6c检测引物、内参基因引物、DNA聚合酶、荧光染料、PCR缓冲液和水混合，离心后，进行检测反应。
优选地，所述离心的转速为5500～6500rpm，例如可以是5500rpm、5600rpm、5700rpm、5800rpm、5900rpm、6000rpm、6100rpm、6200rpm、6300rpm、6400rpm或6500rpm，优选为6000rpm。
优选地，所述离心的时间为55～65s，例如可以是55s、56s、57s、58s、59s、60s、61s、62s、63s、64s或65s。
优选地，所述检测反应的程序为：
预变性：94～98℃孵育8～12min，孵育的温度例如可以是94℃、94.5℃、95℃、95.5℃、96℃、96.5℃、97℃、97.5℃或98℃，孵育的时间例如可以是8min、8.5min、9min、9.5min、10min、10.5min、11min、11.5min或12min；
循环扩增：
94～98℃孵育15～30s，孵育的温度例如可以是94℃、94.5℃、95℃、95.5℃、96℃、96.5℃、97℃、97.5℃或98℃，孵育的时间例如可以是15s、16s、17s、18s、19s、20s、21s、22s、23s、24s、25s、26s、27s、28s、29s或30s；
55～65℃孵育30～60s，孵育的温度例如可以是55℃、56℃、57℃、58℃、59℃、60℃、61℃、62℃、63℃、64℃或65℃，孵育的时间例如可以是30s、35s、40s、45s、50s、55s或60s；
65～75℃孵育0.5～5min，孵育的温度例如可以是65℃、66℃、67℃、68℃、69℃、70℃、71℃、72℃、73℃、74℃或75℃，孵育的时间例如可以是0.5min、1min、1.5min、2min、2.5min、3min、3.5min、4min、4.5min或5min；
形成熔解曲线：
93～98℃孵育10～30s，孵育的温度例如可以是93℃、93.5℃、94℃、94.5℃、95℃、95.5℃、96℃、96.5℃、97℃、97.5℃或98℃，孵育的时间例如可以是10s、11s、12s、13s、14s、15s、16s、17s、18s、19s、20s、21s、22s、23s、24s、25s、26s、27s、28s、29s或30s；
58～63℃孵育30～90s，孵育的温度例如可以是58℃、58.5℃、59℃、59.5℃、60℃、60.5℃、61℃、61.5℃、62℃、62.5℃或63℃，孵育的时间例如可以是30s、35s、40s、45s、50s、55s、60s、65s、70s、75s、80s、85s或90s。
优选地，所述循环扩增的次数为30～45次，例如可以是30次、31次、32次、33次、34次、35次、36次、37次、38次、39次、40次、41次、42次、43次、44次或45次，优选为40次。
优选地，所述检测反应过程中升温和降温的速率为1.5～1.8℃/s，例如可以是1.5℃/s、1.6℃/s、1.7℃/s或1.8℃/s。
优选地，所述检测反应前还包括对第一链cDNA进行稀释的步骤。
第五方面，本申请提供了一种第一方面所述的Cox6c检测引物、第二方面所述的Cox6c检测试剂盒、第三方面所述的Cox6c表达水平的检测方法或第四方面所述的检测装置中的任意一种或至少两种的组合在制备神经退行性疾病诊断试剂和/或筛选神经退行性疾病治疗药物中的应用。
本申请中，Cox6c与机体的呼吸作用及能量代谢密切相关，而以阿尔兹海默症为代表的神经退行性疾病中常伴随葡萄糖代谢较低的状态，与线粒体呼吸链存在重要关联，因此，Cox6c的表达水平与神经退行性疾病的发病进程存在一定的关联，可应用于诊断试剂的制备及相关药物的筛选中。
相比于现有技术，本申请具有如下有益效果：
(1)本申请所述Cox6c检测引物特异性好，灵敏性高，用于Cox6c的相关检测中；含有所述引物的Cox6c检测试剂盒可对细胞、组织和器官等多种样本进行定性或定量的检测，应用范围较广；所述Cox6c表达水平的检测方法操作简单，可在大部分实验室中进行操作，以内参基因的表达量作为标准，可以检测样本中Cox6c的相对表达水平，准确度高，灵敏度高，且耗时较短，检测成本较低；
(2)Cox6c基因的表达水平与机体中的能量代谢及AD病程相关，野生型小鼠中，3月龄小鼠和6月龄小鼠的大脑皮层、海马体、脑神经突触和外周血单个核细胞中Cox6c的表达水平无明显变化，而转基因AD小鼠不同组织在不同时间的Cox6c的表达量则发生显著性变化，在大脑皮层、海马体和外周血单个核细胞中表达量降低，在脑神经突触中表达量升高，RNA-seq和基因功能富集的结果也显示Cox6c与神经退行性疾病相关，因此可应用于神经退行性疾病诊断试剂制备和相关药物筛选的工作中，与常规的药物筛选工作相比，本申请所述方法成本更低，用时更短，操作也更为简便，具有广阔的应用前景。
附图说明
图1A为本申请实施例1中3月龄野生型小鼠和转基因AD小鼠的基因差异分布的火山图；
图1B为本申请实施例1中6月龄野生型小鼠和转基因AD小鼠的基因差异分布的火山图；
图1C为本申请实施例1中野生型小鼠和转基因AD小鼠的差异显著基因的 热图；
图1D为本申请实施例1中差异显著基因在疾病相关的信号通路上富集的结果图片；
图2A为本申请实施例4中3月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的大脑皮层(Cortex)和海马体(Hippocampus)中的Cox6c的相对表达水平的检测结果图片；
图2B为本申请实施例4中3月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的脑神经突触(SD)中的Cox6c的相对表达水平的检测结果图片；
图3为本申请实施例5中3月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的外周血单个核细胞(PBMCs)在细胞刺激剂佛波脂(PMA)刺激后的Cox6c的相对表达水平的检测结果图片。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
原料：
转基因AD小鼠购自Jackson实验室；
TRIzol购自Invitrogen；
OptiPrep梯度离心剂购自Thermo Fisher Scientific；
组织匀浆平衡液为自行配制，为终浓度为320mmol/L的蔗糖溶液、10mmol/L的三(羟甲基)氨基甲烷盐酸盐溶液、1mmol/L的乙二胺四乙酸溶液、2.5mmol/L的二硫苏糖醇溶液和0.25mmol/L的氟化钠溶液的混合溶液，其中，蔗糖、三(羟甲基)氨基甲烷盐酸盐、乙二胺四乙酸、二硫苏糖醇和氟化钠均购自Sigma-Aldrich；
分离液购自Sigma-Aldrich；
细胞洗涤液购自Sigma-Aldrich；
反转录反应试剂购自Thermo Fisher Scientific；
检测反应试剂购自Thermo Fisher Scientific；
DPBS购自Gibco；
RNA-seq服务由诺禾致源公司提供；
100×的PMA混合液购自Sigma-Aldrich；
RPMI-1640购自Gibco。
实施例1
本实施例使用RNA-seq技术对3月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的脑神经突触(SD)中差异性表达的基因进行分析，对比野生型和AD型小鼠之间的差异，筛选错误发现率(FDR，false discovery rate)<0.01的基因，对基因相对表达差异在2倍以上的基因进行基因富集分析(GO分析)，结果如图1A、图1B和图1C所示。
图1A为3月龄野生型小鼠和转基因AD小鼠的基因差异，图1B为6月龄野生型小鼠和转基因AD小鼠的基因差异，从中挑选出表达量显著性差异的基因，做成热图，如图1C所示，可知共有12个基因的表达量具有显著差异，其中包括Cox6c。与野生型小鼠进行比较可知，3月龄转基因AD小鼠的脑神经突触中Cox6c的表达量低于野生型小鼠，随着AD的发生发展，6月龄转基因AD小鼠的脑神经突触中Cox6c的表达量显著升高。
对上述表达显著的基因进行基因功能富集分析(GO分析)，结果如图1D和表1所示。
表1
通路 功能描述 FDR
Mmu05012 帕金森症 3.10e-08
Mmu00190 氧化磷酸化 3.10e-08
Mmu0510 阿尔兹海默症 3.63e-08
Mmu0516 亨廷顿式病 4.72e-08
Mmu0310 核糖体相关 5.03e-07
由图1D和表1可知，Cox6c的功能主要富集于神经退行性疾病及能量代谢的相关过程中，说明Cox6c的表达水平与神经退行性疾病的发展相关。
综合上述实验结果，可知在神经退行性疾病的进程中，Cox6c的表达变化对病程具有指示作用，因而在神经退行性疾病诊断试剂及相应药物的筛选中具有广阔的应用前景。
实施例2
本实施例提供Cox6c检测引物，所述引物的序列如SEQ ID No.1～2所示：
SEQ ID No.1：GGGAAGGACGTTGGTGTAGA；
SEQ ID No.2：CCAGCAATATGAACCCGCAG。
所述Cox6c检测引物特异性强，灵敏度高，扩增产物的大小为111bp，可用于Cox6c的定性或定量的检测中。
实施例3
本实施例提供一种Cox6c检测试剂盒，所述检测试剂盒包括反转录试剂和检测反应试剂。
所述反转录试剂包括Oligo dT、随机引物、反转录酶、dNTPs、RNA酶抑制剂、反转录缓冲液和无RNA酶污染的去离子水；所述检测反应试剂包括Cox6c检测引物SEQ ID No.1～2、内参基因GAPDH的扩增引物SEQ ID No.3～4、内参基因HPRT的扩增引物SEQ ID No.5～6、由DNA聚合酶、SYBR和PCR缓冲液组成的混合溶液和无RNA酶污染的去离子水。
SEQ ID No.3：TCAACAGCAACTCCCACTCTTCCA；
SEQ ID No.4：ACCCTGTTGCTGTAGCCGTATTCA；
SEQ ID No.5：GGAGTCCTGTTGATGTTGCCAGTA；
SEQ ID No.6：GGGACGCAGCAACTGACATTTCTA。
本申请所述Cox6c检测试剂盒中包含了Cox6c检测实验中所必需的试剂，既可以用于定性检测，又可以用于定量检测，可以检测细胞、组织或器官等多种样本，应用范围较广。
实施例4
本实施例使用实施例3制备的Cox6c检测试剂盒，检测3月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的大脑皮层(Cortex)、海马体(Hippocampus)和脑神经突触(SD)中的Cox6c的表达水平，具体步骤如下：
(1)收集样本，并提取RNA；
将3月龄和6月龄同窝野生型和转基因AD小鼠用异氟烷气体麻醉后，断颈后用剪刀迅速断头处死，将头放于冰上，迅速分离小鼠的大脑皮层和海马体。将分离到的组织在含有4U/mL的蛋白酶抑制剂和RNA酶抑制剂的DPBS中洗涤两次。
从大脑皮层中分离脑神经突触。将取出的脑组织置于冰上，在含有蛋白酶抑制剂和RNA酶抑制剂的组织匀浆平衡液中去除软脑膜及血管组织，取大脑皮层置于2mL的组织匀浆平衡液中匀浆，用40μm的组织过滤器过滤组织匀浆液，去除大的组织碎片；
将过滤后的组织匀浆液在4℃下10000×g离心10min，收集沉淀；
将沉淀悬浮于35％的OptiPrep的组织匀浆平衡液中，后置于装有9％、12.5％、15％、25％和35％的OptiPrep梯度离心剂的离心管中，在4℃下10000×g梯度离心24min，小心收集位于9％～12.5％界面之间的悬浮液，即含有脑神经突触的悬液；
收集的脑突触体悬液在4℃下6000×g离心30min，所得沉淀物即为脑神经突触。
将样本在液氮中磨碎，按每100mg组织加入1mL TRIzol的比例，用匀浆仪进行匀浆处理；
将匀浆样本在15℃下放置5min，使核酸蛋白复合物完全分离；
以每使用1mL TRIzol加入0.2mL氯仿的比例，混合后剧烈振荡15s，室温放置3min；
在4℃下10000×g离心15min，把水相转移到新管中；
用异丙醇沉淀水相中的RNA，以每使用1mL TRIzol加入0.5mL异丙醇的比例，室温放置10min；
在4℃下10000×g离心10min，去上清；
用75％乙醇溶液洗涤RNA沉淀，以每使用1mL TRIzol加入1mL75％乙醇溶液的比例，在4℃下7500×g离心5min，去上清；
将RNA沉淀在室温放置5min干燥，加入25μL无RNA酶污染的去离子水溶解RNA。
(2)反转录反应：
以步骤(1)所得总RNA作为模板进行反转录反应，在无RNA酶污染的 0.2mL离心管中配置反应混合物I，组分及添加量如表2所示。
表2
Figure PCTCN2020137129-appb-000001
将反应混合物I混合后离心5s，在70℃孵育5min后，冰浴5min，再向反应混合物I中加入反应混合物II，所述反应混合物II的组分及添加量如表3所示。
表3
组分 体系
5×反转录缓冲液 4μL
RNA酶抑制剂 1μL
10mM dNTPs 2μL
反转录酶 1μL
总体系 8μL
混合后进行反转录反应，程序为：
25℃孵育5min；
42℃孵育60min；
70℃孵育5min，得到第一链cDNA。
(3)检测反应
以步骤(2)所得第一链cDNA作为模板，检测相应样本的Cox6c的表达水平，具体步骤如下：
将第一链cDNA进行10倍稀释，并配置反应体系，如表4所示。
表4
Figure PCTCN2020137129-appb-000002
Figure PCTCN2020137129-appb-000003
将反应体系混合，在6000rpm下离心1min，进行检测反应：
所述检测反应的程序为：
预变性：95℃孵育10min；
循环扩增：95℃孵育15s；60℃孵育60s；70℃孵育1min；
形成熔解曲线：95℃孵育15s；60℃孵育60s；
循环次数为40次；
所述检测反应过程中升温和降温的速率为1.6℃/s。
(4)对检测反应的结果进行统计学分析。
分别检测同一样本中Cox6c、GAPDH和HPRT的表达量，以GAPDH和HPRT的表达量作为标准，计算Cox6c相对内参基因的相对表达量，对实验结果进行统计。数据使用GraphPad Prism软件进行数据处理，统计学方法采用T检验，以P<0.05为差异具有统计学意义。
月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的大脑皮层(Cortex)、海马体(Hippocampus)和脑神经突触(SD)中的Cox6c的表达水平如图2A和图2B所示。
由图2A可知，转基因AD小鼠在大脑皮层和海马体的Cox6c的相对表达量低于野生型小鼠；在野生型小鼠的大脑皮层和海马体中，3月龄小鼠与6月龄小鼠的Cox6c的相对表达量无明显变化，而转基因AD小鼠的大脑皮层和海马体中，Cox6c在6月龄小鼠中的相对表达量显著低于3月龄小鼠；
由图2B可知，3月龄的转基因AD小鼠在脑神经突触中的Cox6c的相对表达量低于野生型小鼠；在野生型小鼠的脑神经突触中，3月龄小鼠与6月龄小鼠的Cox6c的相对表达量无明显变化，而转基因AD小鼠的脑神经突触中Cox6c在6月龄小鼠中的相对表达量显著高于3月龄小鼠，与RNA-seq及GO分析的结果一致。
3月龄转基因AD小鼠对应病人的病程发展为即将迈入轻微智力损伤(MCI) 阶段，而6月龄转基因AD小鼠则对应病程为出现明确阿尔兹海默症特征的阶段，上述结果表明Cox6c的表达水平与AD密切相关，其表达水平的改变对AD的病程具有指示作用。
实施例5
本实施例使用实施例3制备的Cox6c检测试剂盒，检测3月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的外周血单个核细胞(PBMCs)在细胞刺激剂佛波脂(PMA)刺激后的Cox6c的表达水平，具体步骤如下：
(1)收集样本，并提取RNA；
将3月龄及6月龄同窝野生型和转基因AD小鼠用异氟烷气体麻醉后，断颈后用剪刀迅速断头处死，收集小鼠的血液样本；
向15mL离心管中加入3mL分离液，在18℃下复温；
将血液样本小心加于分离液的液面上，在18℃下400×g离心20min；
用吸管吸除分离液上层0.5cm的上清液部分；
小心吸取分离液层及淋巴细胞层，加入一个新离心管中，加入10mL细胞洗涤液混和，在250×g离心10min；
弃上清，使用5mL细胞洗涤液重悬细胞，在250×g离心10min；
弃上清，重复重悬、离心的步骤1次；
使用0.5mL的RPMI-1640重悬细胞，即为外周血单个核细胞。
将外周血单个核细胞以1×10 6/mL的密度接种于细胞培养6孔板中，培养24h后，用100×的PMA处理细胞。刺激24h后收取细胞，进行RNA提取。
RNA提取的步骤与实施例4中的提取步骤相同。
(2)反转录反应：
与实施例4中的反转录反应的步骤相同。
(3)检测反应
与实施例4中的检测反应的步骤相同。
(4)对检测反应的结果进行统计学分析。
与实施例4中的统计学分析的步骤相同。
月龄(3M)和6月龄(6M)的野生型小鼠(WT)和转基因AD小鼠(AD)的外周血单个核细胞(PBMCs)在PMA刺激下的Cox6c的表达水平如图3所 示。
由图3可知，转基因AD小鼠在外周血单个核细胞中Cox6c的表达量低于野生型小鼠；野生型小鼠的外周血单个核细胞中，3月龄小鼠与6月龄小鼠的Cox6c的相对表达量无明显变化，而转基因AD小鼠的外周血单个核细胞中，Cox6c在6月龄小鼠中的相对表达量显著低于3月龄小鼠。这表明Cox6c的表达水平与AD进程相关。
综合图2A、图2B和图3的结果，可知在转基因AD小鼠的大脑皮层、海马体、脑神经突触和外周血单个核细胞中，Cox6c的表达水平低于野生型小鼠；野生型小鼠在3月龄和6月龄时不同组织的Cox6c的表达水平无明显变化，而转基因AD小鼠的Cox6c的表达水平则发生了显著的变化，在大脑皮层、海马体和外周血单个核细胞中表达量降低，在脑神经突触中表达量升高，这表明Cox6c与AD相关，并且Cox6c的表达水平与AD的病程直接相关，因此可将Cox6c的表达水平作为AD的生物标志物，在AD诊断试剂及相关药物的筛选中发挥作用。
综上所述，本申请提供了一种Cox6c的检测引物，所述引物的特异性好，灵敏度高；含有所述引物的Cox6c检测试剂盒可对多种样本进行检测，操作简单，用时较短，应用范围广泛；机体的Cox6c的表达水平与神经退行性疾病的病程密切相关，因此，所述Cox6c的检测引物和所述Cox6c检测试剂盒可以应用于神经退行性疾病诊断试剂的制备及相应药物的筛选中，应用价值极高。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (14)

  1. Cox6c检测引物，其包括SEQ ID No.1～2所示的核酸序列；其中
    SEQ ID No.1：GGGAAGGACGTTGGTGTAGA；
    SEQ ID No.2：CCAGCAATATGAACCCGCAG。
  2. 一种Cox6c检测试剂盒，其包括权利要求1所述的Cox6c检测引物。
  3. 根据权利要求2所述的Cox6c检测试剂盒，其中，所述Cox6c检测试剂盒还包括反转录反应试剂和检测反应试剂。
  4. 根据权利要求3所述的Cox6c检测试剂盒，其中，所述反转录反应试剂包括反转录引物、反转录酶、dNTPs、RNA酶抑制剂和反转录缓冲液。
  5. 根据权利要求4所述的Cox6c检测试剂盒，其中，所述反转录引物包括Oligo dT和/或随机引物。
  6. 根据权利要求3-5中任一项所述的Cox6c检测试剂盒，其中，所述检测反应试剂包括DNA聚合酶、荧光染料和PCR缓冲液；
    任选地，所述荧光染料包括SYBR；
    任选地，所述Cox6c检测试剂盒还包括去离子水，所述去离子水包括无RNA酶污染的去离子水。
  7. 根据权利要求2-6中任一项所述的Cox6c检测试剂盒，其中，所述Cox6c检测试剂盒还包括内参基因引物；
    任选地，所述内参基因引物包括GAPDH的扩增引物和/或HPRT的扩增引物；
    任选地，所述GAPDH的扩增引物包括SEQ ID No.3～4所示的核酸序列；
    任选地，所述HPRT的扩增引物包括SEQ ID No.5～6所示的核酸序列。
  8. 一种Cox6c表达水平的检测方法，其包括使用权利要求1所述的Cox6c检测引物和/或使用权利要求2～7任一项所述的Cox6c检测试剂盒，检测样本中的Cox6c的表达水平，并进行统计学分析。
  9. 根据权利要求8所述的Cox6c表达水平的检测方法，其中，所述检测样本中的Cox6c的表达水平包括反转录反应和检测反应；
    其中所述反转录反应包括：
    将模板、反转录引物和水混合，孵育后进行冰浴，得到反应混合物I；
    向所述反应混合物I中加入反转录酶、dNTPs、RNA酶抑制剂和反转录缓冲液，进行反转录反应，得到第一链cDNA；
    任选地，所述模板包括RNA模板；
    任选地，所述混合后还包括离心的步骤，所述离心的时间为3～5s；
    任选地，所述孵育的温度为65～75℃，优选为70℃；
    任选地，所述孵育的时间为3～7min，优选为5min；
    任选地，所述冰浴的时间为不少于2min；
    任选地，所述反转录反应的程序为：
    23～28℃孵育4～6min；
    40～45℃孵育55～65min；
    68～72℃孵育3～7min。
  10. 根据权利要求9所述的Cox6c表达水平的检测方法，其中，所述检测反应包括：
    将第一链cDNA、Cox6c检测引物、内参基因引物、DNA聚合酶、荧光染料、PCR缓冲液和水混合，离心后，进行检测反应；
    任选地，所述离心的转速为5500～6500rpm，优选为6000rpm；
    任选地，所述离心的时间为55～65s；
    任选地，所述检测反应的程序为：
    预变性：94～98℃孵育8～12min；
    循环扩增：94～98℃孵育15～30s；55～65℃孵育30～60s；65～75℃孵育0.5～5min；
    形成熔解曲线：93～98℃孵育10～30s；58～63℃孵育30～90s；
    任选地，所述循环扩增的次数为30～45次，优选为40次；
    任选地，所述检测反应过程中升温和降温的速率为1.5～1.8℃/s；
    任选地，所述检测反应前还包括对第一链cDNA进行稀释的步骤。
  11. 根据权利要求8-10任一项所述的Cox6c表达水平的检测方法，其中，在所述检测样本中的Cox6c的表达水平前还包括获取样本并提取RNA的步骤。
  12. 根据权利要求8-11任一项所述的Cox6c表达水平的检测方法，其中，所述样本包括神经突触和/或外周血单个核细胞。
  13. 根据权利要求8-12任一项所述的Cox6c表达水平的检测方法，其中，所述检测方法具体包括以下步骤：
    (1)收集神经突触和/或外周血单个核细胞样本，并提取RNA；
    (2)反转录反应：
    将RNA模板、Oligo dT和/或随机引物和水混合，离心3～5s，在65～75℃下孵育3～7min后，冰浴至少2min，得到反应混合物I；
    向所述反应混合物I中加入反转录酶、dNTPs、RNA酶抑制剂和反转录缓冲液，进行反转录反应，程序为：
    23～28℃孵育4～6min；
    40～45℃孵育55～65min；
    68～72℃孵育3～7min；
    通过上述反应，得到第一链cDNA；
    (3)检测反应：
    对第一链cDNA进行稀释，将稀释后的第一链cDNA、Cox6c检测引物、内参基因引物、DNA聚合酶、荧光染料、PCR缓冲液和水混合，5500～6500rpm离心55～65s，进行检测反应；
    所述检测反应的程序为：
    预变性：94～98℃孵育8～12min；
    循环扩增：94～98℃孵育15～30s；55～65℃孵育30～60s；65～75℃孵育0.5～5min；
    形成熔解曲线：93～98℃孵育10～30s；58～63℃孵育30～90s；
    所述循环扩增的次数为30～45次；
    所述检测反应过程中升温和降温的速率为1.5～1.8℃/s；
    (4)对检测反应的结果进行统计学分析。
  14. 权利要求1所述的Cox6c检测引物、权利要求2-7任一项所述的Cox6c检测试剂盒或权利要求8-13任一项所述的Cox6c表达水平的检测方法中的任意一种或至少两种的组合在制备神经退行性疾病诊断试剂和/或筛选神经退行性疾病治疗药物中的应用。
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