CN109266751B - Biomarker combination for nasopharyngeal carcinoma diagnosis and application - Google Patents

Abstract

The invention discloses a biomarker combination for nasopharyngeal carcinoma diagnosis and application thereof, and relates to the technical field of tumor diagnosis. The biomarker combination disclosed by the invention comprises the following microRNAs: has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730. The biomarker combination is used for diagnosing nasopharyngeal carcinoma and has good sensitivity and specificity.

Description

Biomarker combination for nasopharyngeal carcinoma diagnosis and application

Technical Field

The invention relates to the technical field of tumor diagnosis, in particular to a biomarker combination for diagnosing nasopharyngeal carcinoma and application thereof.

Background

Nasopharyngeal carcinoma (NPC) is a cancer caused by nasopharyngeal epithelial cells, which is very rare in most parts of the world, and the incidence rate is less than 1 year per 10 ten thousand people. However, it is quite common in south china, southeast asia and north africa. From a demographic trend, men are three times more likely to develop the disease than women, with peak ages between 50 and 60 years. Nasopharyngeal carcinoma is also highly associated with Epstein-Barr virus (EBV) infection, genetic susceptibility, smoking and drinking, and environmental factors. Radiotherapy is often an effective treatment for patients with early stages of nasopharyngeal carcinoma, while the prognosis for patients with advanced stages is often poor. Thus, early detection is beneficial to improve the cure in NPC patients.

With the rapid development of the molecular pathogenesis of nasopharyngeal carcinoma, many biomarkers related to diagnosis and prognosis have been reported, including EBV DNA, circulating micrornas (miRNAs), miRNAs of EBV virus, certain cytokines, and several methylated genes. However, the detection of these molecules relies primarily on invasive samples, such as fresh or formalin-fixed paraffin-embedded (FFPE) tissue, plasma or serum, which are prone to patient discomfort. Previous studies reported that specific molecules in body fluids may be closely related to the function of surrounding tissues. Saliva is an easily accessible body fluid near the nasopharyngeal tissue and is a promising non-invasive sample for detecting biomarkers of nasopharyngeal cancer. Molecules in the tissue and plasma may also be present in saliva due to the exchange of substances in the circulation of blood and tissue, and the large blood supply of the blood to the salivary glands. Therefore, the molecules in saliva can be used for detecting systemic diseases of human body, especially diseases of tissues near salivary glands.

MicroRNA (miRNA) is an endogenous non-coding RNA with the length of about 18-25nt, has a plurality of important regulation functions in cells, and is widely considered to be closely related to the occurrence of human diseases. Evidence suggests that mirnas play important roles in tumorigenesis similar to oncogenes or tumor suppressor genes, and can be used in tumor diagnosis and therapy monitoring. mirnas are stable in vitro, and studies suggest that mirnas are secreted and released into blood mainly by cells in circulation, exist in the form of exosomes (exosomes) or protein complexes, and can resist digestion by rnases and harsh conditions, so that biological information is transmitted between cells. Specific mirnas or miRNA combinations in serum or plasma have been reported in the literature for use in the detection of various cancers. However, research on the application of the miRNA molecules in saliva to the detection of cancer markers is slow, and the application of the miRNA molecules in saliva to the detection of nasopharyngeal carcinoma is not reported.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a biomarker combination for diagnosing nasopharyngeal carcinoma, which can be used for diagnosing the nasopharyngeal carcinoma and has better sensitivity and specificity.

It is another object of the present invention to provide a reagent for detecting the above biomarker combination, which can detect the biomarker in a sample.

Another object of the present invention is to provide the use of the above-mentioned agents.

The invention also aims to provide a nasopharyngeal carcinoma diagnosis kit and a nasopharyngeal carcinoma diagnosis chip.

The invention is realized by the following steps:

in one aspect, the present invention provides a biomarker combination for nasopharyngeal carcinoma diagnosis, comprising the following microRNA molecules (miRNA molecules):

has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730;

wherein the nucleotide sequence of the has-miR-30b-3p is shown in SEQ ID NO. 1; the nucleotide sequence of the has-miR-1202 is shown in SEQ ID NO. 4; the nucleotide sequence of has-miR-1321 is shown in SEQ ID NO. 7; the nucleotide sequence of has-miR-3612 is shown in SEQ ID NO. 10; the nucleotide sequence of has-miR-4478 is shown in SEQ ID NO. 13; the nucleotide sequence of the has-miR-4730 is shown in SEQ ID NO. 16.

The research of the invention discovers that the microRNA molecules with differential expression in saliva of nasopharyngeal carcinoma patients and normal controls, namely has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730. And experiments verify that the several microRNA molecules are used as biomarkers for diagnosing nasopharyngeal carcinoma, so that not only individual microRNA molecules show good specificity and sensitivity (fig. 2-7), but also the combination (or understanding as combination) of the microRNA molecules shows better specificity and sensitivity (fig. 8). Based on the above, the microRNA molecules, namely any one or combination of more of has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730, can be used as a biomarker for diagnosing nasopharyngeal carcinoma, and reagents, such as primers or probes, for detecting the microRNA molecules can be used for preparing a nasopharyngeal carcinoma diagnostic kit or chip.

In another aspect, the present invention provides a reagent for detecting the above biomarker combinations.

Further, in some embodiments of the invention, the reagent is a probe having a sequence complementary to the microRNA molecule;

or the reagent is a primer for detecting the microRNA molecules.

Further, in some embodiments of the invention, the primers comprise reverse transcription primers, quantitative PCR pre-primers, and quantitative PCR post-primers;

preferably, a reverse transcription primer sequence for detecting has-miR-30b-3p is shown as SEQ ID NO.2, a primer sequence before quantitative PCR is shown as SEQ ID NO.3, and a primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

preferably, a reverse transcription primer sequence for detecting has-miR-1202 is shown as SEQ ID NO.5, a primer sequence before quantitative PCR is shown as SEQ ID NO.6, and a primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

preferably, the reverse transcription primer sequence for detecting has-miR-1321 is shown as SEQ ID NO.8, the primer sequence before quantitative PCR is shown as SEQ ID NO.9, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

preferably, the reverse transcription primer sequence for detecting has-miR-3612 is shown as SEQ ID NO.11, the primer sequence before quantitative PCR is shown as SEQ ID NO.12, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

preferably, the reverse transcription primer sequence for detecting has-miR-4478 is shown as SEQ ID NO.14, the primer sequence before quantitative PCR is shown as SEQ ID NO.15, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

preferably, the reverse transcription primer sequence for detecting has-miR-4730 is shown as SEQ ID NO.17, the primer sequence before quantitative PCR is shown as SEQ ID NO.18, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21.

On the other hand, the invention provides the application of the reagent in preparing a nasopharyngeal carcinoma diagnostic kit or a nasopharyngeal carcinoma diagnostic chip.

Further, in some embodiments of the present invention, the detection sample of the nasopharyngeal carcinoma diagnosis kit is saliva.

In another aspect, the present invention provides a nasopharyngeal carcinoma diagnosis kit, which comprises the above-mentioned reagent.

Further, in some embodiments of the invention, the kit further comprises one or more of the following components:

reverse transcriptase, buffer, dNTPs and Mgcl₂、ddH₂O, fluorescent dye, Taq enzyme, standard substance and contrast.

In another aspect, the invention provides a nasopharyngeal carcinoma diagnosis chip, wherein a miRNA probe for detecting the biomarkers is fixed on the chip.

Further, in some embodiments of the invention, the miRNA probe has a sequence complementary to the microRNA molecule.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Figure 1 is an expression profile of 12 differentially expressed mirnas in example 1.

FIG. 2 is a ROC curve for the has-miR-30b-3p miRNA molecule of example 2.

FIG. 3 is a ROC curve for the has-miR-1202 miRNA molecule of example 2.

FIG. 4 is a ROC curve for the has-miR-1321 miRNA molecule of example 2.

FIG. 5 is a ROC curve for the has-miR-3612 miRNA molecule of example 2.

FIG. 6 is a ROC curve for the has-miR-4478 miRNA molecule of example 2.

FIG. 7 is a ROC curve for the has-miR-4730 miRNA molecule of example 2.

FIG. 8 is a ROC curve for the combination of 6 miRNAs (has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478, and has-miR-4730) in example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Chip screening experiment for differential expression of miRNA in saliva

1. After informed consent was obtained, 10 saliva samples of patients with primarily confirmed nasopharyngeal carcinoma and 10 saliva samples of healthy persons as normal controls were collected from Jiangsu tumor Hospital. Saliva was sampled 1 hour after meal, and 2ml of the saliva was stored in a refrigerator at-20 ℃.

2. RNA was extracted from saliva of nasopharyngeal carcinoma patients and normal persons using the miRNeasy Serum/Plasma Kit (#217184, Qiagen, Hilden, Germany), and the procedures were performed according to the manual with some modifications.

(1) Thoroughly mixing 2ml of the collected saliva sample with 4ml of QIAzol lysate, and standing at room temperature for 5 minutes;

(2) adding 1.1ml of chloroform, thoroughly mixing, and standing at room temperature for 5 minutes;

(3) centrifuging at 4 ℃ for 15 minutes under the condition of 12000 g;

(4) sucking out about 2ml of supernatant, adding 100% ethanol (3ml) with 1.5 times of volume, and mixing gently;

(5) adding the mixed solution into a centrifugal column, adding 700 mul each time, centrifuging at room temperature for 15 seconds under the condition of 8000g, and pouring out waste liquid;

(6) repeating the operation of the step (5), and centrifuging the mixed solution for multiple times;

(7) adding 700 μ l of RWT to the centrifugal column, centrifuging at 8000g for 15 s at room temperature, and removing the waste liquid;

(8) adding 500 μ l of RPE into a centrifugal column, centrifuging at room temperature for 15 seconds under the condition of 8000g, and pouring out waste liquid;

(9) adding 500 μ l of 80% ethanol (ready for use), centrifuging at 8000g for 2 min, and removing waste liquid;

(10) putting the centrifugal column into a new collecting pipe, opening the cover, and centrifuging at full speed for 5 minutes at room temperature by a centrifuge for spin-drying the organic solvent on the centrifugal column membrane;

(11) the column was placed in a new 1.5ml collection tube, 27. mu.l of RNase-free water was added to the center of the membrane, the centrifuge was centrifuged at full speed for 2 minutes at room temperature, 25. mu.l of the solution (with a dead volume of 2. mu.l) in which the RNA was dissolved was collected, and the solution was stored in a freezer at-20 ℃ for further use.

3. In the discovery experiments, one can optionally select a human Unitag miRNA expression profiling chip detection platform to analyze differentially expressed miRNAs in a sample. The chip contains 2017 human miRNAs from Sanger database v.19.0.

(1) Adding 20 μ l of the extracted RNA sample into 80 μ l of hybridization solution (5 XSSC, 0.2% SDS, 100nM special fluorescent report probe, RNase-free water to 80 μ l), and blowing, beating and mixing;

(2) add 100. mu.l of the above liquid to the UnitagTM miRNA chip, cover with 4-well Agilent siliconized coverslip (Agilent, G2534-60011), and place in the chip hybridization box;

(3) the hybridization cassette was placed in a hybridization oven (Agilent, 2545A) for overnight hybridization at 44 ℃ at 15rpm, typically for 16 hours;

(4) after hybridization, the chip and the cover plate were first opened in 400ml of 37 ℃ 5 XSSC solution, then washed 2 times for 3 minutes in 400ml of 37 ℃ 5 XSSC solution containing 0.02% SDS, followed by 2 times for 3 minutes in 400ml of 27 ℃ 0.2 XSSC solution, all four times in total, by rinsing the liquid on a shaker at 100 rpm;

(5) taking the chip out of the washing liquid, and spin-drying on a slide centrifuge;

(6) the chip was scanned with a LuxScan 10K two-channel laser scanner (CapitalBio Inc.), and the chip image was analyzed by LuxScan 3.0 image analysis (CapitalBio Inc.) for data extraction, and the image signal was converted into a digital signal.

4. After background signals are subtracted from the gene chip data, Quantile normalization is carried out on the data in R language, SPSS19.0 software is used for carrying out pairing t-test to obtain a differential expression gene with p being less than 0.01 and difference being more than 2 times, and Cluster 3.0 is used for carrying out Cluster analysis.

5. 10 nasopharyngeal carcinoma patients and normal control saliva are subjected to differential expression analysis by using a miRNA chip, and 12 differentially expressed miRNAs are screened out, namely has-miR-30b-3p, has-miR-575, has-miR-650, has-miR-937-5p, has-miR-1202, has-miR-1203, has-miR-1321, has-miR-3612, has-miR-3714, has-miR-4259, has-miR-4478 and has-miR-4730, and are all expressed in nasopharyngeal carcinoma saliva in a down-regulated manner compared with the normal control (figure 1).

Example 2

qRT-PCR experiments of miRNA in saliva

1. And according to the miRNA chip result, selecting 6 miRNAs with signal values larger than 500 in the chip result, and carrying out further experiments by using a qRT-PCR method. The primers for the selected has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478, and has-miR-4730 are shown in Table 1 below. The qRT-PCR detection of miRNA is carried out on single saliva individuals of nasopharyngeal carcinoma patients and normal controls, strict quality control is implemented in the whole research, and each sample is continuously detected for three times.

TABLE 1

2. After informed consent was obtained, 80 saliva samples of patients diagnosed with nasopharyngeal carcinoma and 80 saliva samples of healthy persons as normal controls were collected from Jiangsu tumor Hospital.

3. RNA was extracted from saliva of nasopharyngeal carcinoma patients and normal persons using the miRNeasy Serum/Plasma Kit (#217184, Qiagen, Hilden, Germany), and the procedures were performed according to the manual with some modifications.

(1) 1ml of the collected saliva sample was thoroughly mixed with 2ml of QIAzol lysate and left at room temperature for 5 minutes;

(2) adding 0.54ml of chloroform, thoroughly mixing, and standing for 5 minutes at room temperature;

(3) centrifuging at 4 ℃ for 15 minutes under the condition of 12000 g;

(4) sucking out about 1ml of supernatant, adding 1.5 times of 100% ethanol (1.5ml), and mixing gently;

(5) adding the mixed solution into a centrifugal column, adding 700 mul each time, centrifuging at room temperature for 15 seconds under the condition of 8000g, and pouring out waste liquid;

(6) repeating the operation of the step (5), and centrifuging the mixed solution for multiple times;

(7) adding 700 μ l of RWT to the centrifugal column, centrifuging at 8000g for 15 s at room temperature, and removing the waste liquid;

(8) adding 500 μ l of RPE into a centrifugal column, centrifuging at room temperature for 15 seconds under the condition of 8000g, and pouring out waste liquid;

(9) adding 500 μ l of 80% ethanol (ready for use), centrifuging at 8000g for 2 min, and removing waste liquid;

(10) putting the centrifugal column into a new collecting pipe, opening the cover, and centrifuging at full speed for 5 minutes at room temperature by a centrifuge for spin-drying the organic solvent on the centrifugal column membrane;

(11) the column was placed in a new 1.5ml collection tube, 20. mu.l of RNase-free water was added to the center of the membrane, the centrifuge was centrifuged at full speed for 2 minutes at room temperature, 18. mu.l of the solution (with a dead volume of 2. mu.l) in which the RNA was dissolved was collected, and the solution was stored in a freezer at-20 ℃ for further use.

4. Reverse transcription was performed using PrimeScript RT Master Mix (Cat # RR037A, TAKARA) and reverse transcription primers according to the manual.

(1) The preparation of the reaction solution was carried out according to the following table to a total volume of 20. mu.l;

composition (I)	Final concentration	Volume of
			5×Reverse Transcription Buffer	1×	4μl
Has U6/microRNA RT Primer(2uM)	1pmol	1μl
			RNA Template	----	5μl
PrimeScript RT Enzyme Mix 1	----	1μl
			DEPC ddH2O		To 20. mu.l

(2) The prepared reaction solution is gently mixed by a pipette and is centrifuged for a short time, then the reaction is carried out for 15 minutes at 37 ℃ and 5 seconds at 85 ℃, and then the reaction solution is placed on ice and is diluted by adding 20 mul of dd H2O for standby.

5. The qPCR reaction was performed using 480SYBR Green I Master (Cat #04707516001, Roche) and the instrument used Roche LightCycler 480 (Roche).

(1) The preparation of the reaction solution was carried out according to the following table to a total volume of 20. mu.l;

composition (I)	Final concentration	Volume of
			2×Real-time PCR Master Mix	1×	10μl
Has U6/microRNA F Primer(10uM)	0.5μM	1μl
			Has U6/microRNA R Primer(10uM)	0.5μM	1μl
cDNA Template	----	1μl
			dd H2O		To 20. mu.l

(2) Centrifuging the prepared reaction solution for a short time, carrying out PCR reaction, and carrying out reaction according to the following procedure;

6. the PCR amplification result is expressed as Ct value, which is the number of cycles in the PCR reaction at which the fluorescence signal reaches the set threshold. The expression level of miRNA in saliva sample can be expressed by equation 2-delta Ct, wherein the delta Ct is Ct^miRNA-Ct^U6。

7. Data processing was performed using SPSS19.0 software, and Mann-Whitney U test was used to compare the differences in expression of salivary miRNA between nasopharyngeal carcinoma patient groups and normal control groups, with p <0.01 considered statistically significant. ROC curve analysis was performed with medcalc15.8 software and sensitivity and specificity were calculated.

8. Data analysis results show that the differential expression of has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730 has significance (p is less than 0.01), and the areas under ROC curves (AUC) of 6 miRNAs are respectively as follows:

has-miR-30b-3p, 0.883 (95% confidence interval, 0.755-0.958), sensitivity 81.82%, specificity 88.00%;

has-miR-1202, 0.865 (95% confidence interval, 0.733-0.947), sensitivity 81.82%, specificity 88.00%;

has-miR-1321, 0.831 (95% confidence interval, 0.693-0.924), sensitivity 77.27%, specificity 80.00%;

has-miR-3612, 0.855 (95% confidence interval, 0.722-0.941), 77.27% sensitivity and 80.00% specificity;

has-miR-4478, 0.823 (95% confidence interval, 0.684-0.919), sensitivity 72.73%, specificity 80.00%;

has-miR-4730, 0.852 (95% confidence interval, 0.718-0.938), sensitivity 72.73%, specificity 92.00% (FIG. 2-7).

The combined AUC values of these 6 miRNAs were 0.941, (95% confidence interval, 0.831-0.989), sensitivity and specificity were 95.45% and 80.00%, respectively (FIG. 8), which are superior to single miRNA.

These results indicate that has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730 are combined to detect nasopharyngeal carcinoma saliva, and the detection has very high sensitivity and specificity for diagnosing nasopharyngeal carcinoma.

Example 3: qPCR kit for diagnosing human nasopharyngeal carcinoma

The above examples show that has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478, and has-miR-4730 have high diagnostic value (high sensitivity and specificity) for nasopharyngeal carcinoma saliva detection, and therefore, a kit for diagnosing human nasopharyngeal carcinoma can be prepared based on has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478, and has-miR-4730. The kit comprises a has-miR-30b-3p primer and a probe; has-miR-1202 primer and probe; has-miR-1321 primer and probe; has-miR-3612 primer and probe; has-miR-4478 primer and probe; has-miR-4730 primer and probe. The primers specifically include reverse transcription primers, quantitative PCR pre-primers, and quantitative PCR post-primers, as shown in Table 1.

Of course, the kit should also include conventional enzymes and reagents required for the corresponding PCR reaction, such as reverse transcriptase, buffer, dNTPs, MgCl₂、ddH₂O, fluorescent dye, Taq enzyme, standard substance and contrast. The design of primers is a matter of routine skill in the art and other sequences can be designed. The kit has the value that only saliva samples are needed, and tissue and blood samples are not needed, so that the kit can be used for diagnosing nasopharyngeal carcinoma.

Example 4: chip kit for diagnosing nasopharyngeal carcinoma

Similarly, chip-based miRNA detection can also be used for diagnosing nasopharyngeal carcinoma saliva. The expression of has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730 can be detected by manufacturing a chip with a small amount of probes, so that the nasopharyngeal darcinoma can be diagnosed on a saliva sample.

1. All designed miRNA probe sequences in the miRNA chip are completely complementary with full-length mature miRNAs detected correspondingly. The miRNAs detected include has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730. In addition, 2 short oligonucleotides were designed, which are not homologous to known miRNA sequences. In order to immobilize the probes to the aldehyde-modified slide surface, polyA of 15 bases was added to the 5 'end of these probe sequences, and modified with the 5' -amino group of C6. Oligonucleotide probes were synthesized in TAKARA and dissolved in EasyArray (TM) spotting solution at a concentration of 20. mu.M. Spotting was repeated 3 times per probe using a SmartArrayTM spotter (Boo Bio Inc.).

2. Saliva total RNA was extracted using Trizol reagent, and small RNA was extracted using Ambion's miRNA Isolation Kit. The small RNAs were labeled using T4RNA ligase labeling technique by mixing 1. mu.g of small RNA with 500ng of 5 '-phosphate-cytidyl-uridyl-cy 3-3' (Dharmacon, Lafayette) and labeling with T4RNA ligase (New England Biolabs). The labeling reaction was carried out at 4 ℃ for 2 hours. Then precipitated with 0.3M sodium acetate and 2.5 volumes of ethanol, washed with ethanol, dried and suspended in 50. mu.l of hybridization buffer containing 5 XSSC, 0.2% SDS.

3. Chip hybridization was performed in an Agilent hybridization cassette (Agilent, G2534A) using an 8-well siliconized coverslip (Agilent, G2534A-60014) which was hybridized overnight at 44 ℃ at 15rpm in a hybridization oven (Agilent, 2545A) for 16 hours.

4. The chip in 37 ℃ 5 x SSC solution open chip and cover plate, with 0.02% SDS 5 x SSC washing solution washing 3 minutes, 2 times, then in 27 ℃ 0.2 x SSC solution washing 3 minutes, 2 times. After completion, the chip was removed from the wash solution and spun on a slide centrifuge.

5. The chip was scanned with a LuxScan 10K scanner (CapitalBio Inc.), and the obtained image was analyzed by LuxScan 3.0 image analysis (CapitalBio Inc.). After background signals are subtracted from the gene chip data, Quantile normalization is carried out, the expression conditions of the miRNAs are determined, the miRNAs are compared with a control sample, and the provided human saliva sample is diagnosed according to a Logistic regression analysis equation.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Suzhou nanotechnology and nano-bionic institute of Chinese academy of sciences

<120> biomarker combination for nasopharyngeal carcinoma diagnosis and application thereof

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Claims (7)

1. The application of a reagent for detecting a biomarker combination in the preparation of a nasopharyngeal carcinoma diagnostic kit is characterized in that a detection sample of the nasopharyngeal carcinoma diagnostic kit is saliva, and the biomarker combination comprises the following microRNA molecules:

has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730;

wherein the nucleotide sequence of the has-miR-30b-3p is shown in SEQ ID NO. 1; the nucleotide sequence of the has-miR-1202 is shown in SEQ ID NO. 4; the nucleotide sequence of has-miR-1321 is shown in SEQ ID NO. 7; the nucleotide sequence of has-miR-3612 is shown in SEQ ID NO. 10; the nucleotide sequence of has-miR-4478 is shown in SEQ ID NO. 13; the nucleotide sequence of the has-miR-4730 is shown in SEQ ID NO. 16.

2. The use according to claim 1, wherein the reagent is a primer for the detection of microRNA molecules.

3. The use of claim 2, wherein the primers comprise reverse transcription primers, quantitative pre-PCR primers, and quantitative post-PCR primers;

the reverse transcription primer sequence for detecting has-miR-30b-3p is shown as SEQ ID NO.2, the primer sequence before quantitative PCR is shown as SEQ ID NO.3, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

the reverse transcription primer sequence for detecting has-miR-1202 is shown as SEQ ID NO.5, the primer sequence before quantitative PCR is shown as SEQ ID NO.6, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

the reverse transcription primer sequence for detecting has-miR-1321 is shown as SEQ ID NO.8, the primer sequence before quantitative PCR is shown as SEQ ID NO.9, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

the reverse transcription primer sequence for detecting has-miR-3612 is shown as SEQ ID NO.11, the primer sequence before quantitative PCR is shown as SEQ ID NO.12, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

the reverse transcription primer sequence for detecting has-miR-4478 is shown as SEQ ID NO.14, the primer sequence before quantitative PCR is shown as SEQ ID NO.15, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21;

the reverse transcription primer sequence for detecting has-miR-4730 is shown as SEQ ID NO.17, the primer sequence before quantitative PCR is shown as SEQ ID NO.18, and the primer sequence after quantitative PCR is shown as SEQ ID NO. 21.

4. The use according to claim 1, wherein the kit further comprises the following components:

reverse transcriptase, buffer, dNTPs, MgCl₂、ddH₂O, fluorescent dye, Taq enzyme, standard and control.

5. The application of a reagent for detecting the biomarker combination in the preparation of a nasopharyngeal carcinoma diagnosis chip is characterized in that a detection sample of the nasopharyngeal carcinoma diagnosis chip is saliva;

the biomarker combinations include the following microRNA molecules:

has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730;

wherein the nucleotide sequence of the has-miR-30b-3p is shown in SEQ ID NO. 1; the nucleotide sequence of the has-miR-1202 is shown in SEQ ID NO. 4; the nucleotide sequence of has-miR-1321 is shown in SEQ ID NO. 7; the nucleotide sequence of has-miR-3612 is shown in SEQ ID NO. 10; the nucleotide sequence of has-miR-4478 is shown in SEQ ID NO. 13; the nucleotide sequence of the has-miR-4730 is shown in SEQ ID NO. 16.

6. The use according to claim 5, wherein the agent is a probe having a sequence complementary to the microRNA molecule.

7. The use of claim 5, wherein said nasopharyngeal carcinoma diagnosis chip is immobilized with a probe for detecting a combination of biomarkers.

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