CN110699457B - Primer group and kit for detecting lung cancer - Google Patents

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a primer group and a kit for detecting lung cancer. The primer group comprises: a first outer primer pair shown as SEQ ID NO.1 and SEQ ID NO.2, and a first inner primer pair shown as SEQ ID NO.3 and SEQ ID NO. 4; a second outer primer pair shown as SEQ ID NO.5 and SEQ ID NO.6, and a second inner primer pair shown as SEQ ID NO.7 and SEQ ID NO. 8; a third outer primer pair shown as SEQ ID NO.9 and SEQ ID NO.10, and a third inner primer pair shown as SEQ ID NO.11 and SEQ ID NO. 12; a fourth outer primer pair shown as SEQ ID NO.13 and SEQ ID NO.14, and a fourth inner primer pair shown as SEQ ID NO.15 and SEQ ID NO. 16.

Description

Primer group and kit for detecting lung cancer

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a primer group and a kit for detecting lung cancer.

Background

The lung cancer is the most common malignant tumor in the world, 210 ten thousand new lung cancers are generated worldwide in 2018, account for 11.6 percent of all new tumor cases, die for 180 ten thousand, account for 18.4 percent of all tumor death cases, and are ranked first. The lung cancer is one of main malignant tumors in China, and due to the large population base, the number of new cases and death cases of the lung cancer in China is far higher than that of other countries, and the disease burden of the lung cancer is always very heavy.

The survival rate of early lung cancer is far higher than that of late lung cancer, and researches show that the one-year survival rate of the first-stage lung cancer can reach 85 percent and is five times higher than that of a fourth-stage lung cancer patient, so the key points for reducing the death rate of the lung cancer patient are early diagnosis and early treatment. In the current lung cancer detection technology, the traditional invasive (invasive) method, such as tissue biopsy, is gradually replaced by the advanced non-invasive (non-invasive) method, wherein the liquid biopsy technology based on biomarker molecule detection has a wide application prospect in the early diagnosis of lung cancer.

Tumor protein biomarkers such as carcinoembryonic antigen (CEA), squamous Cell Carcinoma Antigen (SCCA), neuron-specific enolase (NSE), cytokeratin 19 fragment (Cyfra 21-1, KRT19) and the like are widely used for screening diagnosis, curative effect evaluation, postoperative detection, prognosis judgment and the like of lung cancer. The utility of these markers has been well established in clinical practice, however, protein molecule detection has great limitations in specificity and sensitivity compared to nucleic acid molecule detection due to unavoidable cross-reactivity in antigen-antibody reaction. Compared with protein molecules, the nucleic acid molecules have the great advantages that the nucleic acid molecules can be amplified through a PCR technology and identified by a sequencing technology, so that the sensitivity and the specificity can be obviously improved.

The current nucleic acid molecular markers for early diagnosis of lung cancer are mainly tumor DNA (ctDNA) and micro ribonucleic acids (miRNA) which are circulated in plasma or contained in exosome. The DNA is used as a tumor marker, and cancer diagnosis is mainly carried out by detecting methylation, nucleotide mutation or gene fusion sites. The methylation step is complicated, the technical barrier is high, and the accuracy is difficult to ensure. However, the randomness of gene mutation or fusion is large, and the positioning of the affected region is difficult. For example, KIF5B-RET, even though two high-frequency genes are fused in lung cancer, has a fusion site whose location in the genome is different. In addition, the currently detected DNA tumor markers change with a low frequency, for example, in one research report, the ALK fusion gene positive rate is only 3.43%, while in another report, the EML4-ALK gene fusion detection rate is 7.3%. It follows that the coverage of the detection means based on DNA molecules is relatively low.

miRNA is widely applied to the development of liquid biopsy as a tumor marker, however, as a non-coding single-stranded RNA molecule with the length of only about 22 nucleotides, the accuracy and specificity of the miRNA are judged by a sequence with certain difficulty. Abcam developed a miRNA-based lung cancer diagnostic kit (product number ab 04060) but stopped promotion, probably because of accuracy and specificity. In addition, the number of the miRNA identified at present is limited, and only about 2300 miRNA exist, so that the application potential of the miRNA in cancer diagnosis is greatly limited. The expression of long-chain RNA such as mRNA and LncRNA can be greatly changed in tumor cells, but at present, the application of liquid biopsy is rarely reported, and the concern of RNA degradation is a main reason. However, studies have shown that mRNA fragments in non-cryogenically long-stored samples can still be efficiently extracted and quantified. In addition, the change of mRNA and LncRNA can more accurately reflect the physiological status of patients than the limited number of mirnas currently identified, and thus, the application of long-chain RNA-related fragments in plasma is receiving increasing attention for cancer diagnosis.

In addition, the existing lung cancer molecular diagnosis method based on quantitative analysis cannot clearly distinguish the pathological changes of the Chronic Obstructive Pulmonary Disease (COPD) and the lung cancer, for example, the lung cancer marker molecule CA125 which is generally adopted in clinic is also up-regulated in the COPD condition actually, so how to distinguish the COPD and the lung cancer is a great challenge in early diagnosis of the lung cancer.

Therefore, the prior art is in need of improvement.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides a primer group and a kit for detecting lung cancer, and aims to solve the technical problems of low specificity and sensitivity and unsatisfactory detection effect of the conventional lung cancer detection.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention provides a primer group for detecting lung cancer, which comprises the following components: a first outer primer pair shown as SEQ ID NO.1 and SEQ ID NO.2, and a first inner primer pair shown as SEQ ID NO.3 and SEQ ID NO. 4; a second outer primer pair shown as SEQ ID NO.5 and SEQ ID NO.6, and a second inner primer pair shown as SEQ ID NO.7 and SEQ ID NO. 8; a third outer primer pair shown as SEQ ID NO.9 and SEQ ID NO.10, and a third inner primer pair shown as SEQ ID NO.11 and SEQ ID NO. 12; a fourth outer primer pair shown as SEQ ID NO.13 and SEQ ID NO.14, and a fourth inner primer pair shown as SEQ ID NO.15 and SEQ ID NO. 16.

In another aspect, the present invention provides a kit for detecting lung cancer, wherein the kit comprises a reverse primer set for detecting lung cancer, and the primer set comprises: a first outer primer pair shown as SEQ ID NO.1 and SEQ ID NO.2, and a first inner primer pair shown as SEQ ID NO.3 and SEQ ID NO. 4; a second outer primer pair shown as SEQ ID NO.5 and SEQ ID NO.6, and a second inner primer pair shown as SEQ ID NO.7 and SEQ ID NO. 8; a third outer primer pair shown as SEQ ID NO.9 and SEQ ID NO.10, and a third inner primer pair shown as SEQ ID NO.11 and SEQ ID NO. 12; a fourth outer primer pair shown as SEQ ID NO.13 and SEQ ID NO.14, and a fourth inner primer pair shown as SEQ ID NO.15 and SEQ ID NO. 16.

The primer group and the kit provided by the invention can assist in diagnosing early lung cancer by detecting the combination of plasma RNA markers based on a real-time fluorescent quantitative PCR technology. The primer group and the kit have strong specificity and high sensitivity for detecting the lung cancer, can overcome the limitation existing in the existing molecular diagnosis of the lung cancer, can sensitively and specifically detect the trace RNA fragment in the blood plasma by using the primer group to carry out real-time fluorescence quantitative PCR, can efficiently judge the occurrence of the lung cancer, are not only used for distinguishing lung cancer patients from healthy people, but also can eliminate the interference of COPD in the diagnosis, have higher specificity and sensitivity compared with the existing clinical auxiliary diagnosis method (such as protein molecular markers), and have wide application prospect.

Drawings

FIG. 1 is a graph showing the effect of different kits in the present invention on plasma RNA extraction;

FIG. 2 is a diagram showing the comparative results of RT-PCR of different nested primers in the present embodiment;

FIG. 3 is a graph showing the influence of the number of primer species on PCR efficiency in pre-amplification in the example of the present invention;

FIG. 4 is a graph of sensitivity results for pre-amplification (5, 10, 15, 20 cycles) versus qPCR in an example of the invention;

FIG. 5 is a graph of the results of the sensitivity effect on qPCR without pre-amplification in an example of the invention;

FIG. 6 is a graph showing the single-marker detection results of the MALAT1 molecular marker in the example of the present invention;

FIG. 7 is a graph showing the results of single index detection of XIST1 molecular markers in the examples of the present invention;

FIG. 8 is a graph showing the single-index detection results of MMP1 molecular markers in the present example;

FIG. 9 is a diagram showing the result of detecting a single index of a CP molecular marker in an embodiment of the present invention;

FIG. 10 is a graph showing the combined diagnosis results of MALAT1/XIST1/MMP1/CP four-molecular marker combinations in the present example; a is a statistical result graph of lung cancer and a healthy group, B is a statistical result graph of lung cancer and COPD + a healthy group, and C is a final derivation result graph.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

It is to be understood that the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, features defined as "first", "second", "third" may explicitly or implicitly include one or more of the features.

MALAT-1 (metallosis associated in lung adeno specific transcript 1, malat 1) lacks significant open coding frame, and cannot translate protein in vitro, and belongs to long non-coding RNA (lncRNA). MALAT-1 is the first long non-coding RNA reported to be associated with lung cancer disease.

The X-inactive specific transcription factor (XIST) is a transcription product of XIST gene located in the center of X-chromosome inactivation, plays an important role in the development of tumor as an oncogene, and the high-expression XIST promotes the development of cancer cells of NSCLC (non-small cell lung cancer).

MMP1 (matrix metalloproteinase 1) is part of the MMP matrix metalloproteinase gene cluster that maps to chromosome 11q22.3. Participates in the decomposition of extracellular matrix, plays an important role in tumor metastasis by decomposing I, II and III type interstitial collagens and can promote the migration of NSCLC cells.

Ceruloplasmin (CP) is Ceruloplasmin or copper oxidase, and the mutation of the CP can cause anaplasmacytoma, iron accumulation and tissue damage, is related to diabetes and nervous system abnormality, and can be used as a prognostic factor for evaluating the recurrence risk of tumors after radical treatment of NSCLC.

The specificity and sensitivity caused by cross reaction in the detection of protein molecular markers are low, the coverage rate existing in the detection of DNA methylation, nucleotide mutation or gene fusion sites is not high, the selection of miRNA tumor markers is less, and the accuracy and specificity cannot be fully ensured. Therefore, in the embodiment of the invention, the four genes are all lung cancer related genes, the lung cancer related genes are used as lung cancer liquid biopsy tumor markers, the solid theoretical basis is provided, a set of MALAT1/XIST1/MMP1/CP four-molecular marker combinations are summarized by detecting the level changes of lung cancer related mRNA and LncRNA in plasma, and the primer group is designed, so that the differences between a healthy group and a lung cancer group and between a COPD group and the lung cancer group can be effectively detected.

Because the plasma RNA yield is low, the cDNA synthesized by reverse transcription is also extremely trace, the target template is not easy to detect by direct qPCR, and the nonspecific PCR background is increased, the nested PCR (nested PCR) method for detecting the target gene expression level with enhanced sensitivity and specificity is adopted in the embodiment. In this example, two pairs of primers (i.e., outer primer pair for pre-amplification: upstream F1 and downstream R1; inner primer pair for qPCR amplification: upstream F2 and downstream R2) were designed for each target gene, and a pair of inner primers for qPCR was first designed, and then a pair of outer primers for pre-amplification was designed according to the positioning of the inner primers.

The design requirements of the primers are as follows: designing an inner primer pair, namely qPCR primer, and meeting the requirements, 1) the length of the primer is 22-24 bases, and the GC content is 45% -55%; 2) PCR product 100-200bp; 3) Spanning at least one intron in the genomic DNA; 3) All known transcription variants (transcription variants) that can cover the target gene; 4) The primer region was designed so that the sequence did not coincide with other genes. The design requirements of the outer primer pair are as follows: 1) The length of the primer is 19-21 basic groups, and the GC content is 45% -55%; 2) The PCR product is 250-300bp and contains an inner primer amplification region.

The final sequence is as follows:

for MALAT-1 gene (NCBI reference number: NR _ 002819.4)

A first outer primer pair for pre-amplification:

1, SEQ ID NO.1:5'-GCATCCGAAGGAATGCTTGA-3' (upstream)

SEQ ID NO.2:5'-GATCCAAGCTACTGGCTGC-3' (downstream)

A first inner primer pair for qPCR amplification:

SEQ ID NO.3:5'-AAAGCAAGGTCTCCCCACAAG-3' (upstream)

SEQ ID NO.4:5'-GGTCTGTGCTAGATCAAAAGGC-3' (downstream)

For XIST gene (NCBI reference number: NR _ 001564.2)

A second outer primer pair for pre-amplification:

SEQ ID No.5:5'-CCTTTCGCATTGCTTCTGAG-3' (upstream)

SEQ ID NO.6:5'-GTGGGCAAGAATGGTTCTTG-3' (downstream)

A second inner primer pair for qPCR amplification:

SEQ ID NO.7:5'-TCAGCCCATCAGTCCAAGATC-3' (upstream)

SEQ ID No.8:5'-CCTAGTTCAGGCCTGCTTTTCAT-3' (downstream)

For MMP1 gene (NCBI reference number: NM-001145938.2)

A third pair of outer primers for pre-amplification:

SEQ ID No.9:5'-GAAGCATATCGATGCTGCTC-3' (upstream)

SEQ ID NO.10:5'-CTTTGGACTCACACCATGTG-3' (downstream)

A third inner primer pair for qPCR amplification:

SEQ ID No.11:5'-GATCTATGGATCCAGGTTATCCC-3' (upstream)

SEQ ID NO.12:5'-GCTATTAGCTTTCTGGAGAGTC-3' (downstream)

For CP gene (NCBI reference number: NM-000096.4)

A fourth pair of outer primers for pre-amplification:

SEQ ID NO.13:5'-AGGTCCACAACTTCATGCAG-3' (upstream)

SEQ ID No.14:5'-CCACTGTAGAGGTCCTTAAC-3' (downstream)

A fourth inner primer pair for qPCR amplification:

SEQ ID NO.15:5'-CCACAAGGCCCTACTCAATACAT-3' (upstream)

SEQ ID No.16:5'-GCCCATGGAATACAAGCAGAATC-3' (downstream)

And in one embodiment, the primer set includes the following fifth outer primer pair and fifth inner primer pair designed for the reference gene beta-actin gene (NCBI reference number: NM-001101.5).

A fifth pair of outer primers for pre-amplification:

SEQ ID NO.17:5'-GTACGCCAACACAGTGCTG-3' (upstream)

SEQ ID NO.18:5'-GCCATGCCAATCTCATCTTG-3' (downstream)

A fifth inner primer pair for qPCR amplification:

SEQ ID No.19:5'-AGGATGCAGAAGGAGATCAC-3' (upstream)

SEQ ID No.20:5'-TGTAACGCAACTAAGTCATAG-3' (downstream).

On the other hand, the embodiment of the invention also provides a kit for detecting lung cancer, the kit comprises a reverse primer group for detecting lung cancer, and the primer group comprises: a first outer primer pair shown as SEQ ID NO.1 and SEQ ID NO.2, and a first inner primer pair shown as SEQ ID NO.3 and SEQ ID NO. 4; a second outer primer pair shown as SEQ ID NO.5 and SEQ ID NO.6, and a second inner primer pair shown as SEQ ID NO.7 and SEQ ID NO. 8; a third outer primer pair shown as SEQ ID NO.9 and SEQ ID NO.10, and a third inner primer pair shown as SEQ ID NO.11 and SEQ ID NO. 12; a fourth outer primer pair shown as SEQ ID NO.13 and SEQ ID NO.14, and a fourth inner primer pair shown as SEQ ID NO.15 and SEQ ID NO. 16.

Furthermore, the primer group in the kit also comprises a fifth outer primer pair and a fifth inner primer pair aiming at the reference gene beta-actin gene.

Further, the kit comprises a plasma RNA extraction reagent. The Plasma RNA extraction reagent is selected from miRNeasy Serum/Plasma Kit. Further, the kit comprises an RNA reverse transcription reagent. The RNA reverse transcription reagent is selected from PrimeScript RT Master Mix, and the RNA reverse transcription reagent contains random hexamer primer and utilizes random hexamerThe primer is subjected to reverse transcription. Further, the kit comprises PCR reagents. The PCR reagent is used for nested PCR amplification, and is specifically selected from

PCR Master Mix and TB Green Taq Mix, pre-amplification and qPCR amplification were performed, respectively. The outer primer pair and the inner primer pair of each target gene form a group of nested primers for pre-amplification and qPCR amplification respectively.

Further, when the first outer primer pair, the second outer primer pair, the third outer primer pair and the fourth outer primer pair are used for amplification, the concentration of the first outer primer pair, the second outer primer pair, the third outer primer pair and the fourth outer primer pair is 0.2um. When the first inner primer pair, the second inner primer pair, the third inner primer pair and the fourth inner primer pair are used for amplification, the concentration of the first inner primer pair, the second inner primer pair, the third inner primer pair and the fourth inner primer pair is 0.2um.

The embodiment of the invention uses the kit to detect the lung cancer, and comprises five steps: plasma RNA extraction, reverse transcription by using a random hexamer primer, pre-amplification PCR by using an outer primer pair, real-time fluorescent quantitative PCR (qPCR) by using an inner primer pair, and data collection and analysis.

The combination of the primer pair and the kit can be used as a non-invasive liquid biopsy means, and the lung cancer is detected based on real-time fluorescence quantitative analysis of mRNA and LncRNA fragments in plasma, so that compared with the current clinical traditional invasive methods such as tissue biopsy, the kit has the great advantages of small damage, low technical barrier, high sensitivity and good specificity; meanwhile, compared with other molecular detection means, the method also has obvious advantages.

1) Compared with tumor protein biomarkers, the embodiment has higher specificity and sensitivity, because the protein biomarker detection based on antigen-antibody reaction can generate unavoidable cross reaction phenomenon.

2) Compared with tumor DNA markers, the method is simple and convenient to operate, high in accuracy, wide in coverage and high in universality. The detection of the DNA marker is mainly used for cancer diagnosis by detecting methylation, nucleotide mutation or gene fusion sites, the methylation detection steps are complicated, the technical barrier is high, the randomness of gene mutation or fusion is high, and the occurrence frequency of certain specific pathological change is low, so the coverage rate of the detection means is low.

3) Compared with a small RNA marker, such as miRNA, the embodiment has high accuracy and specificity, and the candidate molecule selection range is large. The length of miRNA is only about 22 nucleotides, and the accuracy and specificity of miRNA are difficult to judge through the sequence. In addition, the number of the miRNA identified at present is limited, and only about 2300 miRNA exist, so that the application potential of the miRNA in cancer diagnosis is greatly limited.

4) Compared with the current lung cancer detection means, the embodiment can also clearly distinguish pathological changes of Chronic Obstructive Pulmonary Disease (COPD) and lung cancer.

Changes in the levels of long-chain RNAs such as mRNA and LncRNA can reflect the existence of tumor cells very accurately, and can be released to plasma for free existence. Long-chain RNA in plasma is degraded to different degrees according to the sequence characteristics, so that the composition combination of mRNA and LncRNA in the plasma is greatly different from that in tissues and cells, and the detection of RNA fragments released by tumor cells is very difficult, and a large amount of screening work is required. In this example, a large amount of data mining work is performed, 50 candidate marker molecules of mRNA and LncRNA highly expressed in lung cancer tissues are searched and summarized from scientific articles and public databases, primers are respectively designed and verified in 95 plasma RNAs (plasma of 40 lung cancer patients, plasma of 40 slow obstructive pulmonary disease (COPD) patients and plasma of 15 healthy people), and a MALAT1/XIST1/MMP1/CP four-molecular marker combination is screened out.

In one embodiment, the DNA is relatively stable in plasma, while RNA is readily degraded, so the relative ratio of RNA to DNA levels is not as high as in tissues or cells. In addition, pre-amplification greatly enhances the sensitivity of PCR, and therefore the effect of genomic DNA on the quantitative results must be considered. In the embodiment of the invention, besides the fact that intron crossing is found as much as possible in the design of the primer to eliminate the influence of DNA, different methods are compared in RNA extraction, and the optimal selection of miRNeasy Serum/Plasma Kit (product number # 217184) of Qiagen company (Qiagen) for extracting Plasma RNA is determined through the comparison of different kits (as shown in figure 1), so that DNA can be completely removed, and the purity of RNA is ensured.

In one example, it was demonstrated that efficient primers were designed such that plasma RNA was degraded to different degrees according to sequence, secondary structure, etc., and that for the same mRNA or LncRNA, the level of fragments remaining in plasma could be different, and thus the efficiency of detecting target RNA molecules by primers in different regions was different. Based on such consideration, for target gene RNA with low plasma level and difficult detection, several sets of nested primers are designed in different regions of each gene, plasma RNA is used for RT-PCR comparison, for example, CP gene is taken as an example, four sets of nested primers are designed, as shown in FIG. 2, finally, one set of nested primers (i.e., SEQ ID NO. 13-16) with the highest efficiency is selected and added into the kit, and other three target genes are similar, so that the optimal nested primers are selected. Meanwhile, the RT-outer primer pair PCR-inner primer pair PCR is carried out on the plasma RNA, and then the obtained PCR product is subjected to sequencing analysis, so that the detection of the plasma free DNA cannot be influenced under the high-sensitivity PCR condition.

The plasma RNA extraction yield and its low level are difficult to detect when quantified by conventional RT-qPCR protocols and are affected by primer dimers and non-specific PCR products. The yield of plasma-extracted RNA samples was low and OD _260nm /OD _230nm The lower ratio (as shown in table 1) indicates a lower purity of the extracted product. Therefore, the invention adds a pre-amplification step in the RT-PCR quantitative detection scheme to achieve the purpose of improving the detection sensitivity and specificity. Meanwhile, sequencing analysis is carried out on a PCR product obtained after RT-outer primer PCR-inner primer PCR is carried out on plasma RNA, and detection of plasma free DNA is not influenced under the high-sensitivity PCR condition.

The aim of improving the detection sensitivity and specificity is fulfilled by adding a pre-amplification step; in addition, the pre-amplification reaction conditions are optimized in the embodiment, in the PCR reaction, the efficiency is reduced and the non-specificity is improved due to the excessive addition of the types of the primer pairs, however, if each target gene is pre-amplified independently, the cost and the working strength of the kit are increased, in the embodiment of the invention, the conditions of the types of the outer primer pairs prepared by the pre-amplification PCR are compared, and as shown in FIG. 3, the pre-amplification of 5 genes can be simultaneously performed in one PCR reaction. And the cycle number of pre-amplification and the dilution times for subsequent reactions are also searched, after pre-amplification, PCR reaction is carried out by using Ceruloplasmin (CP) inner primer pairs, and the condition that the obtained pre-amplification PCR product is diluted by 50 times and is used for real-time fluorescent quantitative PCR (qPCR) is determined.

Pre-amplification significantly improves the sensitivity of qPCR. The results in fig. 4 and 5 show the effect on qPCR results before and after the addition of the pre-amplification step. Without pre-amplification, the qPCR lysis curve (across curve) shows only non-specific heterobands, e.g. primer dimers. After the pre-amplification is increased, along with the increase of the pre-amplification cycle number (cycle), a dissolution curve shows that the specific product of qPCR is increased, and the non-specific miscellaneous band gradually disappears, which indicates that the pre-amplification enriches the quantity of the target gene and provides conditions for qPCR detection. The pre-amplification results in FIG. 4 also show that 15 cycles are the best conditions, too low to completely eliminate non-specific hybridization bands, and too high may excessively increase the template amount of qPCR, resulting in large errors in the quantification process.

The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.

Example 1 plasma RNA extraction

This example uses the miRNeasy Serum/Plasma Kit (product # 217184) from Qiagen to extract human Plasma RNA.

1. Plasma samples were separated. After blood draw, EDTA was used as an anticoagulant to avoid hemolysis, and plasma was separated by centrifugation at 2800g for 10min over 2 hours and stored in a-80 ℃ ultra-low temperature refrigerator.

2. Plasma samples were removed from a-80 ℃ ultra-low temperature refrigerator and thawed on ice.

3. The RNA is extracted by 50-200 ul. Plasma RNA was mixed with 5 plasma volumes of Qiazol lysate, vortexed until no significant precipitation occurred, and incubated at room temperature for 5min.

4. Centrifugation was carried out at 12000g for 10min at 4 ℃. The supernatant was pipetted into a fresh 1.5ml sterile EP tube.

5. One plasma volume of chloroform was added, shaken vigorously for 15s, and incubated at room temperature for 2-3min.

6. At 4 ℃ 12000g, centrifuge for 15min.

7. The upper aqueous phase was transferred to a new 1.5ml (sterile) EP tube (prepared with 200ul plasma, about 600ul of upper aqueous phase could be aspirated).

8. Adding 1.5 times of anhydrous ethanol (900 ul of anhydrous ethanol is added into 600ul of supernate) to the volume of the upper water phase, repeatedly blowing, beating and mixing.

9. Sucking 700ul of the mixed liquid to an RNA separation column, centrifuging the column at room temperature (15-25 ℃) for 15s under the centrifugal force of 10000g, discarding the lower liquid layer, and centrifuging the column for 15s under an empty tube.

10. And (4) repeating the operation of the step 10 until the water phase-ethanol mixed solution is completely passed through the column.

11. 700ul of RNA washing Buffer I (Buffer RWT) was added to the above RNA separation column, the column was centrifuged at 10000g for 15s at room temperature (15-25 ℃), the lower layer liquid was discarded, and the column was centrifuged for 15s in an empty tube.

12. 500ul of RNA washing Buffer II (Buffer RPE) was added to the above RNA separation column, the column was centrifuged at a centrifugal force of more than 10000g at room temperature (15-25 ℃) for 15 seconds, the lower liquid was discarded, and the column was centrifuged for 15 seconds in an empty tube.

13. 500ul of RNA washing Buffer II (Buffer RPE) was added to the above RNA separation column, and the column was centrifuged at 10000g for 15s at room temperature (15-25 ℃), and the lower layer liquid was discarded and centrifuged again for 15s in an empty tube.

14. 500ul 80% ethanol was added to the RNA separation column, and the column was centrifuged at 10000g of centrifugal force at room temperature (15-25 ℃) for 2min, and the lower layer liquid and the collection tube were discarded.

15. The column was loaded into a new 2ml collection tube, centrifuged at maximum centrifugal force for 5min, the filter membrane in the column was dried, and the effluent and outer collection tube were discarded.

16. The column was placed in a fume hood, the lid opened, dried for 15min, and the filter was thoroughly dried.

17. The column was transferred to a RNase-free 1.5ml EP tube (supplied in the kit), 15ul RNase-free H2O (ensuring RNase-free H2O was directly applied to the column matrix) was added, allowed to stand at room temperature for 3min, and centrifuged at 12000g for 1min.

18. The tubes containing the RNA were placed on an ice-box for future use and 1ul was taken and quantified using a NanoDrop One UltraQuantum UV Spectrophotometer.

Thirteen RNA samples were extracted as described above and the data are shown in Table 1.

TABLE 1

Example 2 RNA reverse transcription

This example used PrimeScript RT Master Mix (product # DRR 036A) from Takara, inc. to perform reverse transcription using random hexamer primers. Since plasma RNA production is very low and polysaccharide molecules in plasma have a great influence, spectrophotometric quantification may not be accurate, and thus the whole RNA product extracted in example 1 was used for the reverse transcription reaction.

The reverse transcription reaction system is shown in Table 2 below, the reverse transcription reaction procedure is shown in Table 3 below, and the final reverse transcribed cDNA was stored in a refrigerator at-20 ℃.

TABLE 2

Composition (A)	Volume of
		5x PrimeScript RT Master Mix	4ul
Plasma RNA	13ul
		RNase-free H2O	3ul
Total	20ul

TABLE 3

Temperature of	Time
		37℃	15min
85℃	5sec
		4℃	∞

Example 3 PCR amplification

1 nested primer (nested primers) design:

the yield of plasma RNA is low, cDNA synthesized by reverse transcription is also extremely trace, a target template is not easy to detect by direct qPCR, and the background of nonspecific PCR is increased, so that the method for detecting the expression level of a target gene adopts a nested PCR (nested PCR) method with enhanced sensitivity and specificity. In this example, two pairs of primers were designed for each target gene, and a pair of inner primers for qPCR was first designed, and then a pair of outer primers for pre-amplification was designed based on the location of the inner primers. The final primers are shown in SEQ ID NO. 1-16.

2, pre-amplification PCR:

the present example uses Takara corporation

PCR Master Mix (product # RR 300A) performed pre-amplification PCR. Five pairs of external primers can be added to each reaction system simultaneously, namely, pre-amplification PCR of five target genes (MALAT 1, XIST1, MMP1, CP, beta-actin, 5 pairs in total) is carried out.

The pre-amplification PCR reaction system is shown in the following table 4, and the pre-amplification PCR reaction program is shown in the following table 5: after pre-amplification was complete, the PCR product was diluted 50-fold and 1ul was taken for qPCR.

TABLE 4

TABLE 5

3. Real-time fluorescent quantitative PCR (qPCR):

this example used TB Green Taq Mix (product # RR 420B) from Takara for fluorescent quantitative PCR (qPCR). The pre-amplified PCR product obtained above was diluted 50-fold for qPCR.

The qPCR reaction system is shown in table 6 below, the qPCR reaction procedure is shown in table 7 below: two multiple wells were run for each reaction; and finally, collecting data and analyzing.

TABLE 6

Composition (I)	Volume of
		TB Green Taq Mix	6.25ul
Upstream primer (F2, 10 uM)	0.25ul
		Downstream primer (R2, 10 uM)	0.25ul
cDNA Pre-amplification product	1ul
		RNase-free H2O	4.75ul
Total	12.5ul

TABLE 7

4. And (3) data analysis:

after the qPCR is finished, the melting curve (melting curve) is checked first, and all abnormal melting peaks are determined to be No Signal (No Signal Available, NA). The Cycle threshold (Ct value) of the "no signal" test wells were each determined to be 40. Data homogenization correction by subtracting beta-actin Ct from Ct of the target gene to obtain deltaCt (dCt), and then using equation 2 ^dCt Calculating the relative expression quantity of the target gene and carrying out statistical analysis. The single index test results show (shown in FIGS. 6-9): MALAT1/XIST1/MMP1/CP four-molecular marker combination each of which can well distinguish lung cancer (NSCL)C) And Healthy (Healthy) group, AUC exceeds 0.9 except MMP1 (0.875), indicating that both sensitivity and specificity are ideal.

The combined diagnosis results of MALAT1/XIST1/MMP1/CP four-molecular marker combination show that the four-molecular combination of the embodiment can be used for well distinguishing lung cancer (NSCLC) from Healthy people (health) and also distinguishing lung cancer (NSCLC) from COPD + Healthy people (Control) samples (as shown in FIG. 10). After data are collected, an equation deduced by software is imported, and when the result is higher than 0.5925, the tested person can be suspected to be the lung cancer disease.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Shenzhen Rieko Biotech Limited

<120> primer set and kit for detecting lung cancer

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcatccgaag gaatgcttga 20

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gatccaagct actggctgc 19

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaagcaaggt ctccccacaa g 21

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggtctgtgct agatcaaaag gc 22

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cctttcgcat tgcttctgag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgggcaaga atggttcttg 20

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcagcccatc agtccaagat c 21

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cctagttcag gcctgctttt cat 23

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaagcatatc gatgctgctc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctttggactc acaccatgtg 20

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gatctatgga tccaggttat ccc 23

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctattagct ttctggagag tc 22

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aggtccacaa cttcatgcag 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccactgtaga ggtccttaac 20

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccacaaggcc ctactcaata cat 23

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gcccatggaa tacaagcaga atc 23

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gtacgccaac acagtgctg 19

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gccatgccaa tctcatcttg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aggatgcaga aggagatcac 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tgtaacgcaa ctaagtcata g 21

Claims (5)

1. The application of a primer group for detecting the expression quantity of four molecular marker combinations of MALAT-1, XIST, MMP1 and CP in plasma RNA in the preparation of a kit for diagnosing early-stage lung cancer is characterized in that the primer group consists of a first external/internal primer pair for detecting MALAT-1 gene based on nested PCR, a second external/internal primer pair for detecting XIST gene, a third external/internal primer pair for detecting MMP1 gene and a fourth external/internal primer pair for detecting CP gene;

wherein, the first outer primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the first inner primer pair is shown as SEQ ID NO.3 and SEQ ID NO. 4; the second outer primer pair is shown as SEQ ID NO.5 and SEQ ID NO.6, and the second inner primer pair is shown as SEQ ID NO.7 and SEQ ID NO. 8; the third outer primer pair is shown as SEQ ID NO.9 and SEQ ID NO.10, and the third inner primer pair is shown as SEQ ID NO.11 and SEQ ID NO. 12; the fourth outer primer pair is shown as SEQ ID NO.13 and SEQ ID NO.14, and the fourth inner primer pair is shown as SEQ ID NO.15 and SEQ ID NO. 16.

2. The use of claim 1, wherein the first, second, third and fourth outer primer pairs are present at a concentration of 0.2um when the first, second, third and fourth outer primer pairs are used for amplification; and/or the presence of a gas in the gas,

when the first inner primer pair, the second inner primer pair, the third inner primer pair and the fourth inner primer pair are used for amplification, the concentration of the first inner primer pair, the second inner primer pair, the third inner primer pair and the fourth inner primer pair is 0.2um.

3. The use of claim 1, wherein the kit comprises a plasma RNA extraction reagent.

4. The use of claim 1, wherein the kit comprises an RNA reverse transcription reagent.

5. The use of claim 1, wherein the kit comprises PCR reagents.

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