CN108300788B

CN108300788B - Micro ribonucleic acid composition for detecting light brain trauma and application thereof

Info

Publication number: CN108300788B
Application number: CN201710026248.9A
Authority: CN
Inventors: 汪俊军; 卜晓敏; 宋佳希; 王成
Original assignee: Nanjing General Hospital of Nanjing Command PLA
Current assignee: Nanjing General Hospital of Nanjing Command PLA
Priority date: 2017-01-13
Filing date: 2017-01-13
Publication date: 2021-07-09
Anticipated expiration: 2037-01-13
Also published as: CN108300788A

Abstract

The invention belongs to the technical field of biology, and discloses a micro ribonucleic acid composition for detecting light brain trauma and application thereof. The miRNA combinations include: miR-103a, miR-219a, miR-422a and miR-520d, and can further comprise one or more of miR-302d, miR-518f and miR-627. The kit contains a probe combination for detecting the miRNA. The miRNA probe combination provided by the invention can be used for detecting light brain trauma, can improve the detection accuracy, improves the low specificity and low sensitivity caused by individual difference which is difficult to overcome by a single marker, and obviously improves the clinical relevance ratio by combining other detection indexes and clinical symptoms of patients, thereby becoming an effective means for diagnosing light brain trauma. The invention adopts noninvasive serum examination, and is convenient for clinical popularization and use.

Description

Micro ribonucleic acid composition for detecting light brain trauma and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a micro ribonucleic acid (miRNA) combination for detecting light brain trauma and application thereof.

Background

Traumatic Brain Injury (TBI), also known as brain trauma or head trauma, is the damage to brain tissue caused by trauma. TBI occurs not only at the moment of injury, but also in a period of several minutes to several days after the injury due to secondary injury, death and disability caused by changes of cerebral blood flow and intracranial pressure¹. According to statistics of different countries in different periods, the incidence rate of TBI is the first place of trauma²Is the first cause of death of teenagers in developed countries. Along with the continuous improvement of the social and economic levels of China, the traffic is high-speedThe popularity of tools, the rapid development of the construction industry, and the emergence of various rapid and irritating physical exercises have led to a continuing increase in the incidence of TBI. Although the overall mortality rate of TBI is reduced from 50% before 30 years to about 30% at present, the quality of life is not improved correspondingly, and great burden is caused to families and society. Of the surviving patients, 10% of the mildly impaired patients remain permanently disabled, while 66% and 100% of the moderate and severe patients can be reached³. In patients with TBI, mild brain injury (mTBI) accounts for approximately 61.7% of TBI, with a higher proportion. Compared with severe brain injury, the mTBI has relatively small injury degree, but has more hidden clinical symptoms, and can leave a plurality of sequelae which are not easy to relieve for a plurality of patients if timely and effective diagnosis and treatment are not obtained. Therefore, the strengthening of the early diagnosis of TBI, especially mTBI patients has important guiding significance for the treatment work of the neurological intensive care; the craniocerebral injury severity is evaluated early through corresponding indexes, and effective treatment and rehabilitation plans are determined to be of great importance for reducing the disability rate of patients and improving the life quality of the patients.

The main indexes which can be used for diagnosing patients with brain trauma at present are as follows: glasgow Coma Scale (GCS), imaging performance (CT and MRI), electroencephalogram, biochemical metabolic indicators, and the like^4,5However, each method has its limitations and sidedness, such as: firstly, GCS scoring is mainly judged according to whether neurobehaviors such as automatic eye opening, command execution, speech, limb movement and the like exist, and is often limited by factors such as sedatives, trachea cannula and the like; CT and MRI are the most commonly used auxiliary examinations after the brain trauma at present, CT can directly evaluate the craniocerebral injury degree from the structure, but the sensitivity and the specificity are lower, MRI can overcome the defects of CT, but the cost is high, and the time consumption is long; (iii) S100 beta (astrocyte protein), NSE (neuron specific enolase), UCH-1 (ubiquitin carboxyl terminal hydrolase) and GFAP (glial fibrillary acidic protein) can be used as biomarkers for diagnosing heavy brain trauma, but diagnosis of light brain trauma needs to be proved^6,7. Therefore, the specific marker for the patient with light brain trauma is explored and found, and the simple, convenient, quick, accurate and specific trauma situation is provided for clinicObservation and index of curative effect judgment have become a very urgent task in front of us.

In recent years, research finds that micro ribonucleic acid (microRNA, miRNA) is a potential marker for clinical diagnosis and treatment. mirnas are a class of non-coding, endogenous single-stranded small ribonucleic acid molecules of about 19 to 24 nucleotides in length that regulate the expression of a target gene at the post-transcriptional level, primarily by fully or partially complementary binding to the non-translated sequence at the 3 m-terminus of the target gene mRNA, resulting in degradation or post-transcriptional translational inhibition of the target mRNA. The human body has more than 1000 kinds of miRNA, and the miRNA can regulate and control 20-30% of gene expression, thereby regulating the processes of growth, development, physiology, pathology and the like of the human body. In recent years, researches show that the serum miRNA has high stability 8 and specificity, is abnormally expressed in various disease states 9, has the characteristic of convenient material acquisition compared with tissue miRNA, and indicates that the serum miRNA can be used as a noninvasive index for disease diagnosis. At present, the research on serum miRNA at home and abroad mainly focuses on tumor diseases from the beginning, and gradually relates to various diseases such as hepatitis, diabetes, coronary heart disease, brain injury and the like 10. Recent studies have found some miRNAs associated with brain trauma, but have mainly stayed to analyze changes in miRNA expression profiles of tissues and cells at the cellular and animal experimental levels 11-14. In 2012, researchers detect changes of serum miRNA expression profiles of rats with craniocerebral injury for the first time, and find that the levels of serum miR-let-7i, miR-122 and miR-340-5p are remarkably increased, wherein miR-let-7i is enriched in 15 in the brain. In 2014, research reports miRNA expression profiles of mouse closed brain trauma, and the research suggests that after craniocerebral injury, some specifically changed miRNAs exist in tissues and serum and are potential TBI markers. However, there is currently no study on serum-specific mirnas of patients with clinical brain trauma, and 2010 reports that some miRNA levels in serum after brain trauma in humans have been changed 16, but the study has fewer patients (only 10), only 3 days of disease course and no corresponding assessment. The subject group finds that miR-93, miR-191 and miR-499 which is remarkably improved after brain trauma of rats is also remarkably improved after brain trauma of human beings and is remarkably related to the severity degree of the brain trauma, and the subject group can be used for diagnosing the brain trauma 17, but does not carry out gene chip analysis. Therefore, the serum specific miRNA of the severe craniocerebral injury patient is further screened in a large sample, the value of the serum specific miRNA on the diagnosis of the cerebral trauma and the monitoring of the disease condition is evaluated, and the miRNA has important significance on the early diagnosis and treatment of the light craniocerebral injury patient.

At present, no specific miRNA combination and detection biochip or kit which are used for detecting and judging the related traumatic craniocerebral injury by utilizing a non-traumatic blood sample which is convenient to take materials are available.

Disclosure of Invention

The invention aims to screen a group of serum miRNAs with obvious expression difference with normal people by researching the specific change of the serum miRNAs of a light brain trauma patient, and prepare a biochip and a kit suitable for clinical diagnosis and treatment by using the miRNA probes, thereby providing a specific intermediate result and a rapid noninvasive detection means for realizing early detection of light brain trauma.

The above object of the present invention is achieved by the following technical solutions:

a combination of mirnas for detecting mild brain trauma comprising the following mirnas: miR-103a, miR-219a, miR-422a and miR-520 d.

The miRNA combination may further include one or more of the following mirnas: miR-302d, miR-518f and miR-627.

A probe combination for detecting miRNA related to light brain trauma is characterized in that the probe combination comprises a combination of probes for detecting the miRNA. Further, the probe combination also comprises one or more of the following probes for detecting the miRNA: miR-302d, miR-518f and miR-627.

The combination of the probes is as follows: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; further, the probe combination also comprises one or more of the following miRNA probes: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO. 7.

The application of any probe combination in preparing a reagent or a tool for detecting light brain trauma.

A test kit for detecting mild brain trauma, comprising a combination of any one of the probes described above; the kit alsoCan comprise Taq enzyme, dNTP, magnesium chloride and PCR buffer (the PCR buffer is general 10 XPCR buffer without Mg²⁺)。

The following is a further description of the invention:

a combination of mirnas for detecting mild brain trauma comprising the following mirnas: miR-103a, miR-219a, miR-422a and miR-520 d. The miRNA combination may further include one or more of the following mirnas: miR-302d, miR-518f and miR-627.

The screening method of the miRNA combination comprises the following steps (shown in figure 1):

(1) collecting mixed serum or plasma of patients with severe brain trauma, and extracting total RNA;

(2) 754 miRNAs in human genome are detected by adopting TaqMan low-density chip (TLDA) (Applied Biosystems as a manufacturer) with high sensitivity, high accuracy and high repeatability, and the miRNAs with obviously increased brain trauma expression are screened out after being compared with a normal control group matched with age and gender;

(3) detecting miRNA with obvious expression difference screened out from TLDA primarily one by one on a light brain trauma specimen by a fluorescent quantitative PCR method (TaqMan probe method);

(4) further, a quantitative PCR method is used for verifying a large batch of heavy and light brain trauma specimens one by one, and a group of miRNA with stable and obvious expression difference is screened out.

(the primary screening of TLDA aims to screen miRNA with significant difference between the group with heavy brain trauma and the normal group, more emphasis is placed on the qualitative and trend; the secondary screening adopts accurate fluorescent quantitative PCR, the content of each miRNA in a single sample is determined, the emphasis is placed on the quantification; the fluorescent quantitative PCR is further accurate quantification on the basis of the primary screening of TLDA, and the two are consistent but the emphasis is different).

Specifically, the screening method comprises the following steps: (1) respectively collecting mixed serum or plasma of a normal group, a group with good curative effect and a group with poor curative effect on severe cerebral trauma, and extracting total RNA; (2) designing primers according to all 754 known mature miRNAs (designing the primers according to the known miRNAs is a technology known by a person skilled in the art and can be carried out by a primer synthesis company, and the sequences of related primers are not repeated) and a fluorescent probe, sequencing and detecting the RNAs, and primarily screening some miRNAs with significant expression difference between a heavy brain trauma treatment effect difference group and a normal control group; (3) the extracted RNA is reversely transcribed into cDNA, the preliminarily screened miRNA is verified by adopting fluorescence quantitative PCR (TaqMan probe method), and a group of miRNA with stable and specific expression is selected.

The detection method used in the present invention may be selected from: one or more of RT-PCR method, TaqMan low-density chip (TLDA) and Real-time PCR method. For example, the method for detecting miRNA molecules in serum comprises the following steps:

(1) serum total RNA was extracted using Trizol reagent (Invitrogen corporation);

(2) obtaining cDNA through RNA reverse transcription reaction;

(3) designing primers according to all known mature miRNA to perform PCR reaction;

(4) carrying out agarose gel electrophoresis on the PCR product;

(5) the results were observed under uv light after EB staining.

The following steps can be further included after the step (5): changes in the amount of miRNA in a patient's serum sample relative to normal serum are detected and compared using the TaqMan probe method.

The invention also provides a probe combination of miRNA for detecting light brain trauma, which comprises the following TaqMan probe combination of miRNA:

the TaqMan probe combination can further comprise one or more of the following miRNA TaqMan probes:

in addition, the invention provides the application of the probe combination in preparing a reagent or a tool for detecting the light brain trauma.

The invention also provides a biochip for detecting light brain trauma, which comprises the following TaqMan probes:

the above biochip may further comprise one or more of the following probes:

in a specific embodiment of the present invention, the kit comprises Taq enzyme, dNTP, magnesium chloride, and 10 XPCR buffer (without Mg)²⁺) And all miRNA TaqMan probes (supplied by ABI corporation, exclusively for miRNA fluorescence quantification) stably present and detectable in normal serum were included.

The mild brain trauma of the invention comprises various brain traumas with various pathological changes caused by trauma. Specifically, the brain trauma may be traumatic epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral contusion and laceration, diffuse axonal injury. The Grossgo score (Glasgow Coma Scale, GCS) >12 points when the light group is admitted, and less than or equal to 8 points when the heavy group is admitted.

The miRNA combination, the corresponding probe combination and the kit containing the probe combination can be applied to detection of light brain trauma.

The invention has the beneficial effects that:

firstly, the serum is easier to obtain than other tissues, so that the cost is saved and the time consumption is short compared with that of CT or MRI, the use of medical personnel is facilitated, and the pain of a patient is relieved;

secondly, miRNA in serum reflects the overall pathological and physiological conditions of the organism, and the detection result has accurate and detailed guiding significance;

thirdly, the detection of serum miRNA can reflect the regulation state of genes after transcription in the disease occurrence process on the molecular level, and provides a potential target spot for the treatment of light brain trauma.

Fourthly, the TaqMan probes contained in the kit are a group of serum miRNA TaqMan probes which are screened out based on TLDA and quantitative PCR technology and have obvious expression difference in light brain trauma and normal state, so that the detection sensitivity and specificity can be increased, and the detection level is improved.

In conclusion, the specific miRNA combination provided by the invention is used as an intermediate result for diagnosing mild brain trauma, and is helpful for early diagnosis of mild brain trauma by a physician in combination with clinical symptoms, medical history or other examination information. The method for detecting the miRNA in the serum is simple and easy to implement and has excellent effect. The biochip and the kit for detecting the serum miRNA are put into practical use, the serum miRNA level of a patient is detected only through the most simplified TaqMan probe library, the detection index of the light brain trauma can be expanded, the detection sensitivity is improved, the means for detecting the brain trauma is enriched, the serum miRNA is expected to become an important marker molecule for early diagnosis of the light brain trauma, and the serum miRNA has extremely important clinical application potential and value.

Drawings

FIG. 1 is a main flow chart of the present invention.

FIGS. 2-8 are ROC curve analyses of 7 selected miRNAs (A-G) in the mild brain trauma and normal control group. 67 cases of mild brain trauma, 66 cases of normal control.

FIGS. 9-15 are ROC curve analyses of 7 selected miRNAs (A-G) in brain trauma and normal control groups. 132 cases of brain trauma (67 cases with mild symptoms and 65 cases with severe symptoms), and 66 cases of normal controls.

Detailed Description

The invention is further illustrated by the following examples.

Example 1: primary screening of specific miRNA expression profile of brain trauma

(1) Respectively collecting 15 cases, 15 cases and 15 cases of serum samples of a heavy poor curative effect group, a heavy good curative effect group and a normal person, and respectively mixing the three groups of samples;

(2) three groups of mixed serum RNA are respectively extracted, and the specific scheme is as follows: extracting total RNA with Trizol reagent (Invitrogen), and purifying RNA from the supernatant by removing impurities such as protein with phenol chloroform;

(3) three sets of pooled serum total RNA were subjected to Taqman low density chip analysis (TLDA).

The specific scheme is as follows: the essence of TLDA is a 384-well fluorescent quantitative PCR technology, PCR amplification primers and fluorescent probes for quantitative detection of specific genes are added into each well, the expression condition of 384 genes in a sample can be detected simultaneously, each chip card is provided with 8 sample adding holes, so each chip can also be used for specificity of the primers and the probes, the detected signal intensity is in direct proportion to the number of DNA molecules, the sensitivity of the obtained result is higher than that of a common chip, the experimental conditions are easy to optimize, and the chip card is particularly suitable for detection of miRNA.

Mu.l of total RNA was added to the reaction tube and placed in a PCR instrument programmed for reverse transcription. The resulting cDNA was placed on ice and preamplified. After pre-amplification, 0.1M Tris-EDTA buffer (pH 8.0) was added, followed by TLDA loading and final machine reaction.

If the Ct values of the miRNAs in the three groups are all less than 30, and compared with the TLDA result of normal serum, the miRNA in the good-outcome group and the miRNA in the poor-outcome group are both increased, and the miRNA content in the poor-outcome group is more than 5 times that in the normal group, the miRNA is regarded as being significantly changed, otherwise, the miRNA is regarded as not changed. As a result, it was found that 12 of the 754 mirnas were significantly elevated in the patient serum. (details are shown in Table 1)

TABLE 1 primary TLDA-screened miRNA's whose expression is upregulated in heavy brain trauma serum

Example 2: real-time fluorescent quantitative PCR experiment (TaqMan probe method) of miRNA in serum

And verifying batch samples one by adopting a quantitative PCR method, and screening miRNA with obvious expression difference from the miRNA primarily screened by the TLDA method.

The extracted serum total RNA is first reverse transcribed into cDNA. For each miRNA, a gene-specific reverse primer containing the same stem-loop structure is designed, and reverse transcription is performed by using the miRNA-specific reverse primer to obtain cDNA (product of ABI) containing a common stem-loop structure but belonging to a specific miRNA.

The ABI Prism 7300 fluorescence quantitative PCR instrument is used as the instrument.

Because of the instability of the common serum endogenous reference gene, some studies find that the exogenous reference gene can well control the difference in the extraction and purification processes of RNA, and the research finds that the difference in the extraction and purification processes of a sample can be well controlled by adding the exogenous plant miRNA MIR2911 (5'-GGCCGGGGGACGGGCUGGGA-3', SEQ ID No.8) in the RNA extraction process. Therefore, the content of miRNA in serum is calculated by a relative quantitative method. And subtracting the Ct value of the reference gene in the corresponding sample from the Ct value of the miRNA to be detected in the sample to obtain a delta Ct value. The content of each miRNA to be detected relative to the reference gene is 2^-ΔCtAnd (4) showing.

Example 3: rescreening the primary screened serum miRNA by a quantitative PCR method

One group of subject samples are detected one by using a TaqMan quantitative PCR method, and miRNA with significant expression difference between a heavy brain trauma treatment effect poor group and a control group are screened from 12 miRNA primarily screened out by TLDA (see example 2 for specific steps). 24 patients with severe brain trauma and 24 patients with mild brain trauma in this group were compared with 24 patients who were normally treated in Nanjing general Hospital, the military region (see Table 2 for detailed clinical information of patients). As a result, the expression quantity of 7 miRNAs in the 12 preliminarily screened miRNAs in a patient group is obviously higher than that of a control group (the content expression method is mean value +/-standard deviation; the light group variation multiple is 2.97-9.08 times, and p is less than 0.0001), and the 7 miRNAs are miR-103a, miR-219a, miR-422a, miR-520d, miR-302d, miR-518f and miR-627 (see Table 3).

TABLE 2 clinical information of the Subjects

TABLE 3 MiRNA rescreened by quantitative PCR method

Example 4: clinical verification of serum miRNA screened by quantitative PCR method

And further verifying 7 kinds of miRNA screened out again in a large-batch clinical specimen by a quantitative PCR method, and screening out stable miRNA with obvious expression difference. Subjects in the clinical study were 132 patients with brain trauma, 65 in the heavy cohort and 67 in the light cohort. Age and sex matched normal 66 cases were used as controls (see table 4 for detailed clinical information in both groups). The verification result proves that the expression quantity of the 7 screened miRNAs in a large batch of patient samples is obviously higher than that of a control (the content expression method is mean value plus or minus standard deviation, the mean value is 2.55-5.26 times of that of a control group, and p is less than 0.001) (the result is shown in a table 5 and figures 9-15).

TABLE 4 basic clinical information on the Subjects

TABLE 5 results of verifying the re-screened bulk samples for miRNA by quantitative PCR

Example 5: evaluation of clinical value of screened miRNA

Combining the serum samples of the rescreened group and the verified group, drawing an ROC curve according to the expression quantity of miRNA in the serum of each sample, and calculating the area under the curve (AUC) to evaluate the accuracy of the screened miRNA on the diagnosis of the light brain trauma (figures 2-8). As a result, the AUC of these 7 miRNAs was found to be between 0.780 and 0.904 (see Table 6). Further indicates that the miRNA combination has higher sensitivity to diagnosis of mild brain trauma.

TABLE 6 AUC of miRNA selected

Example 6: kit for detecting serum miRNA

The preparation process and the operation flow of the serum miRNA kit for detecting the light brain trauma are based on the TLDA technology and the quantitative PCR technology, and the following TaqMan probes are combined: SEQ ID NO: 1-4 or SEQ ID NO: 1-4 and SEQ ID NO: 5-7, respectively collecting one or more of the obtained products into a PCR kit (RT-PCR or Real-time PCR) to prepare a lightweight brain trauma detection kit with a specific TaqMan probe combination.

The specific composition of the kit (detection of each miRNA) is as follows:

the specific operation flow of the prepared kit is as follows:

(1) collecting a serum sample of a subject, extracting RNA, and performing reverse transcription to prepare a cDNA sample;

(2) the samples are added according to the formula, various miRNAs are respectively measured, and during the measurement, a TaqMan probe specific to each miRNA is used.

(3) The PCR reaction was carried out under conditions of 95 ℃ for 5min, 95 ℃ for 15s, and 60 ℃ for 1min (the stage of 60 ℃ for 1min comprising two steps of annealing and extension, which is a program recommended by ABI), for 40 cycles.

The kit comprises the serum miRNA probe provided by the invention, Taq enzyme, dNTP and other reagents. The kit has the value that the specific TaqMan probe combination is used for detecting the expression quantity of the serum miRNA, so that the sensitivity and the accuracy of the detection of the light brain trauma can be improved, and the early detection of the disease is facilitated. Therefore, the kit is put into practical use, and diagnosis and treatment of diseases can be pushed to a new height.

Reference documents:

1.McNair ND.Traumatic brain injury[J].The Nursing clinics of North America,1999,34(3):637-659

2.Andersson EH,Bjorklund R,Emanuelson I,et al.Epidemiology of traumatic brain injury:a population based study in western Sweden[J].Acta neurologica Scandinavica,2003,107(4):256-259

3. zhang Xiao, Zhang Hao, Indoma research progress [ J ] Chinese rehabilitation theory and practice, 2008,14(2):101-

4.Oliveira RA,Araujo S,Falcao AL,et al.Glasgow outcome scale at hospital discharge as a prognostic index in patients with severe traumatic brain injury[J].Arquivos de neuro-psiquiatria,2012,70(8):604-608

5.Giacoppo S,Bramanti P,Barresi M,et al.Predictive biomarkers of recovery in traumatic brain injury[J].Neurocritical care,2012,16(3):470-477

6.Papa L,Lewis LM,Falk JL,et al.Elevated levels of serum glial fibrillary acidic protein breakdown products in mild and moderate traumatic brain injury are associated with intracranial lesions and neurosurgical intervention[J].Annals of emergency medicine,2012,59(6):471-483

7.Goyal A,Failla MD,Niyonkuru C,et al.S100b as a prognostic biomarker in outcome prediction for patients with severe traumatic brain injury[J].Journal of neurotrauma,2013,30(11):946-957

8.Mitchell PS,Parkin RK,Kroh EM,et al.Circulating microRNAs as stable blood-based markers for cancer detection[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(30):10513-10518

9.Chen X,Ba Y,Ma L,et al.Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases[J].Cell research,2008,18(10):997-1006

10.Jones CI,Zabolotskaya MV,King AJ,et al.Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma[J].British journal of cancer,2012,107(12):1987-1996

11.Liu L,Sun T,Liu Z,et al.Traumatic brain injury dysregulates microRNAs to modulate cell signaling in rat hippocampus[J].PloS one,2014,9(8):e103948

12.Miao W,Bao TH,Han JH,et al.Voluntary exercise prior to traumatic brain injury alters miRNA expression in the injured mouse cerebral cortex[J].Brazilian journal of medical and biological research＝Revista brasileira de pesquisas medicas e biologicas/Sociedade Brasileira de Biofisica...[et al.],2015,48(5):433-439

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Claims

1. The application of a probe combination for detecting a serum-derived miRNA combination in preparing a reagent or a tool for detecting light brain trauma is disclosed, wherein the miRNA combination comprises serum-derived miR-103a, miR-219a, miR-422a and miR-520 d.

2. The use according to claim 1, characterized in that the combination of mirnas further comprises one or more of the following serum-derived mirnas: miR-302d, miR-518f and miR-627.

3. Use according to claim 1, characterized in that said combination of probes is: SEQ ID number 1, SEQ ID number 2, SEQ ID NO.3 and SEQ ID number 4.

4. Use according to claim 3, characterized in that the probe combination further comprises one or more of the following probes: SEQ ID number 5, SEQ ID number 6, SEQ ID number 7.