CN116121453A

CN116121453A - Improved structure of high-sensitivity fluorescent PCR detection primer and probe and application thereof

Info

Publication number: CN116121453A
Application number: CN202211410397.2A
Authority: CN
Inventors: 李红玉; 颜蓉; 韩勋领
Original assignee: Chongqing Puji Life Technology Co ltd
Current assignee: Chongqing Puji Life Technology Co ltd
Priority date: 2022-11-11
Filing date: 2022-11-11
Publication date: 2023-05-16

Abstract

The invention provides a high-sensitivity fluorescent PCR detection primer and probe improved structure and application thereof, belonging to the technical field of gene detection. The improved primer structure of the invention comprises four parts from 5 'to 3': the first part and the third part are in reverse complementary pairing; the sequences of the second part and the fourth part are complementarily matched with the target gene sequence. The improved probe structure of the present invention comprises five parts from the 5 'to the 3' end: the sequences of the first part and the third part are complementarily matched with the sequence to be detected of the target gene; the sequences of the second part and the fourth part are in reverse complementary pairing, and the fifth part is identical with the sequence of the third part. The primer and probe improved structure is used for the PCR detection of human SDC2 gene methylation, novel coronavirus ORF1ab gene and N gene, has the advantages of high sensitivity and good specificity, and can be used as a high-sensitivity detection system for accurately detecting trace mutation, ultra-low content pathogenic microorganisms, excessive interference DNA and the like in tumor liquid biopsy.

Description

Improved structure of high-sensitivity fluorescent PCR detection primer and probe and application thereof

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a primer and probe improved structure for high-sensitivity fluorescent PCR detection, a detection system and application thereof, which are mainly used for fluorescent PCR detection of trace mutation of non-diagnostic tumor liquid biopsy, ultra-low content pathogenic microorganisms and excessive interference DNA.

Background

The PCR technology is one kind of nucleic acid in vitro amplification technology and has the basic principle of simulating the natural DNA copying process in vitro, and has high sensitivity, high specificity, simple operation and other features. However, the method can only perform qualitative analysis or semi-quantitative analysis on the product, cannot accurately quantify the product, and is easy to cause pollution, false positive and other problems when the product identification analysis is complex.

The real-time fluorescent quantitative PCR technology is taken as a second generation PCR technology, and is a technology of adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in real time by utilizing fluorescent signal accumulation, and quantifying nucleic acid by different fluorescent detection methods. The technology realizes the leap of PCR from qualitative to quantitative, is not easy to generate pollution, and has the advantages of higher sensitivity, specificity and accuracy, accurate quantitative, real-time monitoring and the like compared with the first-generation PCR. However, the quantitative result is calculated by comparing the quantitative result with a reference substance, and the quantitative result still belongs to the category of relative quantification depending on a Ct value, a standard curve and a reference sample, and has the defects of insufficient sensitivity and false negative in the aspects of detection of an extremely small amount of nucleic acid sample, specific detection in the presence of an interfering substance, rare mutation detection and small difference identification of the expression quantity.

Scientific researchers at home and abroad have carried out a great deal of researches on specific primers and probes for real-time fluorescence quantitative PCR detection, and developed a series of primers and probes for detecting various nucleic acids. Compared with the beginning of the application of the technology, with the continuous deep research, the structure of the primer and the probe for PCR detection is continuously improved, and the specificity and the sensitivity for PCR detection are also continuously improved. However, the problem of low sensitivity still exists in aspects of trace mutation of tumor liquid biopsy, excessive existence of ultra-low content pathogenic microorganisms and interference DNA and the like.

Colorectal cancer is one of three most common malignant tumors, has the characteristics of high morbidity, high fatality rate and the like, seriously threatens the life and health of patients, strengthens colorectal cancer screening and discovers a proper and effective screening method, and has important significance in improving the detection rate of early tumors and reducing the fatality rate. Colonoscopes, while being the gold standard for screening colorectal cancers, require cumbersome and time-consuming preparation of the intestinal tract, and present a risk of bleeding and perforation as an invasive examination means, with a low participation rate in the screened population.

Extensive research on ^[1] It has been shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression; and are widely found in a variety of tumors. In addition, study ^[2] The stool DNA methylation level change is closely related to colorectal cancer occurrence and development, abnormal methylation often occurs in early colorectal tumor, and the method has high accuracy in detection of near-far colorectal cancer (CRC) and large adenoma (with the diameter more than or equal to 1 cm). Therefore, the human SDC2 gene in the fecal sample of the colorectal cancer high risk group is taken as a target gene, and the methylation level of the human SDC2 gene is detected, so that the human SDC2 gene can be used as a tumor marker for early screening of colorectal cancer.

Numerous scholars have conducted a great deal of research on primers and probes for detecting SDC2 gene methylation in human feces, and a series of important results are obtained, and although the detection sensitivity of gene methylation is effectively improved, the detection sensitivity still cannot meet high requirements in the face of lower methylation mutation proportion and lower DNA sample content.

Patent document CN 109943638A ^[3] A primer probe combination for detecting SDC2 gene methylation is disclosed, which has better specificity for detecting colorectal cancer, however, the primer probe combination can only complete detection of 200 ng-2 mug for an extracted DNA sample, and the detection sensitivity of the primer probe combination needs to be further improved for a lower amount of DNA sample.

Patent document CN 110373470a ^[4] Disclosed is a colorectal tumor specific methylationThe detection primer, the detection probe and the detection kit comprise a forward primer, a reverse primer and a detection probe of the SDC2 gene, wherein ACTB is selected as an internal reference gene, so that the sensitivity of methylation detection of the SDC2 gene is improved, however, the total quantity of qualified genome DNA in a sample detected by the method needs to be more than 1 mug, the detection limit of methylation of the SDC2 gene still cannot be increased to be below ng level, and the trace mutation of the gene cannot be accurately detected.

Patent document CN 114008219A ^[5] Disclosed is a method for detecting methylation of SDC2 gene by using a primer for specifically amplifying a methylated SDC2 gene and a probe capable of complementary hybridization with the methylated SDC2 gene which has been specifically amplified by the primer, which enables accurate detection of 20ng of template DNA but fails to detect accurately in the presence of a lower amount of template DNA.

Patent document CN 110904228A ^[6] Disclosed are a colorectal cancer auxiliary diagnosis kit for fecal nucleic acid detection and a use method thereof, wherein the method is used for SDC2 gene methylation detection, can detect 10 ng/. Mu.L of DNA, and effectively improves the detection limit.

Patent document CN 113454243A ^[7] Disclosed are a composition, a kit and an application for colorectal cancer detection, which can be used for detecting colorectal cancer by detecting the methylation state of SDC2 genes, wherein the detection amount of standard DNA is 10ng.

Patent document CN 110699437A ^[8] The kit realizes that enzyme digestion reaction and fluorescence PCR reaction are sequentially carried out in a tube to detect the methylation state of the SDC2 gene by using methylation sensitive restriction enzyme and a marked twin primer, improves the sensitivity, and can detect the methylation of the SDC2 gene when the DNA content is as low as 1ng and the methylation DNA proportion is as low as 1 percent.

Primary application of emulsion PCR technology in construction of ssDNA library and P16 gene methylation and HBV DNA detection ^[9] The emulsion PCR method is adopted to detect gene methylation, and the result shows that:compared with MSP, the two-step emulsion PCR has higher sensitivity and stronger anti-interference capability, and under the interference of unmethylated genes, the two-step emulsion PCR has the following ratio of methylation to unmethylation of 1:100, whereas MSP can only detect methylation at a ratio of methylation to unmethylation of 1:10. The sensitivity of the method to methylation detection in the presence of more unmethylated interfering genes remains to be further improved.

Park et al ^[10] A total of 190 intestinal lavage (BLF) samples were reported to be collected from patients with colorectal tumors and healthy normal individuals. 14 polypectomy specimens were obtained during colonoscopy, bisulphite pyrosequencing assays were performed and methylation specific polymerase chain reactions were quantified to measure SDC2 methylation in tissues and BLFDNs, and the results showed that in the SDC2 methylated BLFDNA test, the sensitivity for detection of CRC and VA was 80.0% and 64.7%, respectively, with a specificity of 88.9%. BLF in patients with multiple tubular adenomas, single tubular adenomas and proliferative polyps showed 62.8%, 26.7% and 28.6% positive for SDC2 methylation, respectively. The method has the detection quantity of 20ng for the gene sample, and the detection sensitivity is required to be improved.

Niu et al ^[11] The methylation status of SDC2 was reported to be detected in cell lines and 398 colorectal tissue samples, and 497 stool samples were further evaluated using real-time methylation-specific PCR, the effect of 17 potential interfering substances on stool methylation SDC2 performance was analyzed, and SDC2 expression was measured. The results show that: the stool test for methylated SDC2 detected 81.1% (159/196) colorectal cancer and 58.2% (71/122) adenoma with a specificity of 93.3% (167/179), of the 17 interfering substances, only high concentrations of berberine inhibited stool detection for methylated SDC2, but the method detected only a concentration of 20 ng/. Mu.L for SDC2 gene.

Liang et al ^[12] A new qPCR detection reagent was used, comprising two differentially methylated regions of SDC2 and CpG, for detection of CRC. Methylation detection of two fragments of the SDC2 gene (SDC 2-a and SDC 2-b) using two primer pairs, the kit can detect methyl groups in fecal DNA samplesChemical SDC2 performs 100% detection, however, only 5-15 ng of DNA sample can be accurately detected.

The above studies show that, although a series of primers and probes and kits have been developed for detecting methylation of SDC2 gene, which shows better specificity and sensitivity, methylation of SDC2 gene can only be detected when the DNA content is as low as 1ng and the proportion of methylated DNA is as low as 1%, but accurate detection cannot be performed on DNA samples with the concentration lower than 1ng, and the detection sensitivity still needs to be further improved.

In addition to the need for accurate detection of DNA methylation for early cancer screening, accurate detection of trace RNAs and mutations thereof is also needed. Therefore, the development of PCR detection specific primers and probes for detecting novel coronavirus genes with ultralow content and high sensitivity is the key point of research at present.

Patent document CN 113151590A ^[13] The invention discloses a novel coronavirus 2019-nCoV ORF1ab, N and E gene detection kit and a preparation and detection method, wherein a multi-target fluorescent PCR detection primer is designed for three conserved genes (1 ab, N and E genes) of 2019-nCoV, wherein the detection lower limit of the ORF1ab gene is 101 copies/mu L, the detection lower limit of the N gene is 101 copies/mu L, and the detection limit of the N gene still needs to be further improved.

Patent document CN 112410465A ^[14] The invention discloses a novel coronavirus SARS-CoV-2 ORF1ab and N gene isothermal amplification primer group and a kit, which respectively design and screen and determine 2 inner primers, 2 outer primers and 2 ring primers as core technologies aiming at each gene, enhance the specificity and sensitivity of a real-time fluorescence isothermal RT-PCR detection kit, and the experimental result shows that the detection sensitivity of the method for the novel coronavirus SARS-CoV-2 ORF1ab gene and N gene is 100 copies/. Mu.L and can not detect samples with the concentration of 10 copies/. Mu.L.

Patent document CN 112662811A ^[15] Discloses a novel coronavirus 4 gene segment multiplex nucleic acid detection kit and application thereof, the invention designs a primer probe combination 1 of a novel coronavirus ORF1ab gene and an N geneAnd primer probe combination 2 of the E gene and the S gene, and meanwhile, 4 nucleic acid targets of the ORF1ab, N, E, S gene are detected, so that the fault tolerance of the nucleic acid detection on virus variation can be improved, two different conditions of virus variation and virus negative can be better distinguished, and the accuracy of new coronavirus infection diagnosis is improved. The detection limit test result of the kit shows that the detection limit of the kit is 100copies/mL, the detection rate under the condition is 100%, and the detection rate at 50copies/mL is 75%.

Zhou Wei, etc ^[16] A SARS-CoV-2 nucleic acid detection kit (product batch number: 2021277) is adopted to carry out fluorescence quantitative PCR detection on the ORF1ab gene and the N gene of the novel coronavirus, the detection rate of weak positive samples is still higher, but the lower detection limit of the kit only reaches 500copies/mL.

Yu et al ^[17] 323 samples of 76 new crown diagnosis patients were subjected to ddPCR and RT-PCR analysis based on two target genes (ORF 1ab and N) and were collected for detection by nasal swabs, pharyngeal swabs, sputum, blood and urine, and the results showed that: of 95 samples positive for both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF 1ab gene, r2=0.83; n gene, r2=0.87). 4 parts (4/161) were detected as negative by RT-PCR, 41 parts (41/67) were detected as single gene positive by ddPCR, and the viral load was 11.1 to 123.2copies per tube, but the total volume detected per tube was not clear in this document.

As is clear from the descriptions of the patent and journal documents, the minimum detection limit of the real-time fluorescence PCR detection technology for the ORF1ab gene and the N gene of the novel coronavirus only reaches 100copies/mL, and the detection technology still needs to be further improved.

Therefore, although the real-time fluorescent PCR technology has the characteristics of good specificity and good detection sensitivity, the distinguishing capability of the mutation of the current probes and primers for PCR detection and the detection capability of the very low amount of virus RNA molecules are still greatly limited. In particular, in aspects of trace mutation, ultra-low content pathogenic microorganisms, excessive existence of interfering DNA and the like of tumor liquid biopsy, the existing probe and primer design method has serious limitation on the effect of PCR detection and lower detection limit sensitivity, so that improvement is needed in aspects of the probe and primer design method.

Reference is made to:

[1]Kadiyska T，NossikoffA.Stool DNA methylation assays incolorectal cancer screening[J].World J Gastroenterol，2015，21(35)：10057-10061.

[2]Ahlquist DA，Zou H，Domanico M，et al.Next-generation stool DNA test accurately detects colorectal cancer and large adenomas[J].Gastroenterology，2012，142(2)：248-256.

[3] CN 109943638A, a primer probe combination for detecting NDRG4, SDC2 and BMP3 gene methylation, application and kit thereof, and the applicant: hunan Baibo Gene technologies Co., ltd., publication date: 2019.06.28.

[4] CN 110373470A, primers, probes and kits for colorectal tumor specific methylation detection, applicant: ajian (forzhou) genemedicine laboratory limited, publication date: 2019.10.25.

[5] CN 114008219A, method for detecting methylation of SDC2 gene, applicant: gene Tuli Co., ltd., publication date: 2022.02.01.

[6] CN 110904228A, a colorectal cancer auxiliary diagnostic kit for fecal nucleic acid detection and a using method thereof, applicant: people and future biotechnology (Changsha) limited, publication date: 2020.03.24.

[7] CN 113454243A, compositions, kits and uses for colorectal cancer detection, applicant: shenzhen Dada Biotechnology (Shenzhen Co., ltd., public days: 2021.09.28.

[8] CN 110699437A, human SDC2 gene methylation detection kit, applicants: proprietary Biotechnology, inc., publication date: 2020.01.17.

[9] the university of Jiangsu doctor paper, "preliminary application of emulsion PCR technology in construction of ssDNA library and P16 gene methylation and HBV DNA detection," authors: shore cocoa, 2017.6.

[10]Park YS，Kim DS，Cho SW，et al.Analysis of syndecan-2 methylation in bowel lavage fluid for the detection of colorectal neoplasm[J].Gut Liver，2018，12:508-515.

[11]Niu Feng，Wen Jialing，Fu Xinhui，et al.Stool DNA Test of Methylated Syndecan-2 for the Early Detection of Colorectal Neoplasia[J].Cancer Epidemiol Biomarkers Prev，2017，26:1411-1419.

[12]Liang Ma，Geng Qin，Fei Gai，et al.A novel method for early detection of colorectal cancer based on detection of methylation of two fragments of syndecan-2(SDC2)in stool DNA[J].BMC Gastroenterology，2022，22:191.

[13] CN 113151590A, novel coronavirus 2019-nCoV ORF1ab, N and E gene detection kit, preparation method and detection method, applicant: chen Qingsen, publication date: 2021.07.23.

[14] CN 112410465A, novel coronavirus SARS-CoV-2 orf1ab and N gene isothermal amplification primer set and kit, applicant: university of ethnic group, publication date: 2021.02.26.

[15] CN 112662811A, a novel coronavirus 4 gene segment multiplex nucleic acid detection kit and application thereof, applicant: beijing Meikang Gene science Co., ltd., public days: 2021.04.16.

[16] zhou Wei, li Junfeng, liu Caizhou, et al, novel coronavirus large-scale nucleic acid screening amplification reagent preservation protocol Industry [ J ]. Gansu science journal, 2022, 34 (4): 34-38.

[17]Fengting Yu，Liting Yan，Nan Wang，et al.Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients[J].CLINICAL INFECTIOUS DISEASES，2020，71(15):793-798.

Disclosure of Invention

The invention aims to solve the technical problems, and provides an improved structure of a primer and a probe suitable for a high-sensitivity fluorescent PCR detection system and application thereof. The technical purpose of the invention is that: solves the problems that the existing probe and primer design is limited in aspects of trace mutation of tumor liquid biopsy, ultra-low content pathogenic microorganisms, excessive interference DNA and the like, such as the detection limit of PCR detection on target gene human SDC2 gene methylation can only be 1 ng/mu L for intestinal cancer patient fecal sample DNA with a mutation proportion of 1 percent, and the detection limit of ORF1ab gene and N gene of the target gene novel coronavirus can only be 100copies/mL, thereby providing a primer and probe design method with high detection sensitivity and good specificity, and a detection method and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a first object of the present invention is to provide an improved primer structure useful for high sensitivity fluorescent PCR detection, comprising a forward primer and a reverse primer;

wherein, the forward primer comprises the following four parts from the 5 'end to the 3' end:

a first part: 4-10 bases in length, and has low sequence homology with human genome sequence;

a second part: the length is 15-20 bases, the sequence of the primer is complementary and paired with a sequence to be detected of a target gene, and the design principle is followed by the design principle of fluorescent quantitative detection primers or Arms-PCR primers;

third section: the sequence of which is complementary to the first part in reverse complement;

fourth part: the length is 3-5 bases, the sequence of the primer is complementary paired with a sequence to be detected of a target gene, and the primer is separated from the 3' of the second part by 2-3 bases;

the first part and the third part of the reverse primer are identical to the forward primer, and the sequences of the second part and the fourth part are respectively complementary and paired with the other sequence of the target gene.

The inventor discovers that the primer structure is designed according to the method, the specificity of the primer can be increased, the sensitivity of the existing PCR detection method is obviously improved after the primer is combined with a probe, the reaction specificity is enhanced, the system stability is improved, and the primer structure can provide a high-sensitivity fluorescent PCR detection system. The improved primer structure of the invention can combine the first part and the third part into double chains under normal conditions or proper conditions (such as room temperature), so that the stability of the primer is improved, and the combination efficiency of the primer and the target fragment is improved by the second part and the fourth part.

Furthermore, in the primer structure provided by the invention, the first partial sequence of the forward primer and the reverse primer is TAACATA, and the third partial sequence is TATATTTA.

Furthermore, the primer structure provided by the invention can be well used for fluorescent PCR detection of human SDC2 gene methylation sequences, novel coronavirus ORF1ab genes and N genes, and has high detection sensitivity after being combined with a probe.

It is a second object of the present invention to provide an improved probe structure for high sensitivity fluorescent PCR detection, the probe comprising the following five parts from the 5 'end to the 3':

a first part: the length is 10-15 bases, the sequence of the probe is complementary and matched with the sequence to be detected of the target gene, and the design principle of the probe is in accordance with the design principle of the probe;

a second part: 4-6 bases in length, and has low sequence homology with human genome sequence;

third section: the length is 10-15 bases, the sequence of the primer is complementarily paired with the sequence to be detected of the target gene, and the primer is separated from the 3' of the first part by 2-3 bases;

fourth part: the sequence of which is complementary to the second part in reverse complement;

fifth part: the sequence is identical to the third partial sequence.

According to the improved probe structure provided by the invention, under normal conditions or under proper conditions (such as room temperature), the second partial sequence and the fourth partial sequence in the probe can be combined into double chains, so that the stability of a reaction system is greatly improved, the background signal is reduced, and the detection sensitivity is improved. In addition, the third partial sequence and the fifth partial sequence of the probe are the same, so that the binding efficiency with the target fragment is improved. The improved probe and primer structure of the invention is used for PCR detection of target genes such as human SDC2 gene methylation, novel coronavirus ORF1ab gene, N gene and the like, and can remarkably improve the detection sensitivity.

Further, in the probe of the present invention, the second partial sequence is GGTAGA and the fourth partial sequence is TCTACC.

Further, the 5 'end of the probe is connected with a fluorescent group, the fluorescent group comprises FAM, VIC, HEX, CY5 or ROX, the 3' end of the probe is connected with a quenching group, and the quenching group comprises TAMRA, BHQ1, BHQ2, MGB or Dabcy1.

Experiments prove that the high-sensitivity detection system composed of the improved primer structure and the improved probe structure has high sensitivity in detection aspects of trace mutation of tumor liquid biopsy, excessive existence of ultra-low content pathogenic microorganisms and interference DNA and the like. Wherein, the PCR detection of the methylation of the target gene human SDC2 gene can realize that the detection limit of the stool sample DNA of the intestinal cancer patient with the mutation ratio of 0.1% is as low as 0.5 ng/. Mu.L, and compared with the existing detection method, the detection sensitivity is improved by more than 20 times. The detection limit of the novel coronavirus ORF1ab gene and the N gene of the target gene reaches 10copies/mL, and compared with the existing detection method, the detection sensitivity is improved by more than 10 times.

When the primer structure and the probe structure of the present invention were changed, as shown in comparative example 1 of the present invention, it was found that the sensitivity of other primer structure and probe structure for PCR detection was significantly reduced, and the 10ng0.5% EGFR gene T790M mutation could not be detected. Therefore, the improved primer structure and probe structure of the invention have high specificity and detection sensitivity in aspects of trace mutation of tumor liquid biopsy, ultra-low content pathogenic microorganisms, excessive existence of interference DNA and the like.

The primer structure and the probe structure provided by the invention can be suitable for fluorescent PCR detection of various objects to be detected, and have high detection sensitivity. The fluorescent PCR detection kit not only can carry out high-sensitivity fluorescent PCR detection on the target genes listed in the examples, but also can have high detection sensitivity on other target genes, and is very suitable for a fluorescent PCR detection system. On the other hand, the high sensitivity detection system can also be applied to ARMS-PCR detection.

It is still another object of the present invention to provide a highly sensitive fluorescent PCR detection system composed of the primer structure and the probe structure as described above, which has high detection sensitivity in PCR detection, such as can be used for highly sensitive detection of human SDC2 gene methylation, novel coronavirus ORF1ab gene or N gene, and it is expected that the detection sensitivity of the primer and probe detection system of the improved structure is extremely high for other genes.

It is a fourth object of the present invention to provide a method for ARMS-PCR detection using the detection system as described above, which, when the primer structure as described above is used, places the mutation site in the fourth part of the primer.

The fifth purpose of the invention is to provide the application of the detection system composed of the primer and the probe structure in PCR detection of trace mutation, ultra-low content pathogenic microorganisms, excessive interference DNA and other aspects for tumor liquid biopsy, and the application is for non-diagnosis purpose.

The beneficial effects of the invention are as follows:

the invention provides an improved primer and probe structure, and a detection system formed by the improved primer and probe structure can have high detection sensitivity and specificity on aspects of trace mutation, ultralow content pathogenic microorganisms, excessive interference DNA and the like of tumor liquid biopsy, and the existing primer and probe design is greatly improved and limited by the detection conditions. The primer and probe structure has high sensitivity and good specificity when used for PCR detection. The detection limit of the target gene human SDC2 gene methylation detection on the fecal sample DNA of the intestinal cancer patient with the mutation ratio of 0.1% can be reduced to 0.5 ng/. Mu.L, and compared with the existing detection method, the detection sensitivity is improved by more than 20 times; the detection limit of the novel coronavirus ORF1ab gene and the N gene of the target gene reaches 10copies/mL, and compared with the existing detection method, the detection sensitivity is improved by more than 10 times.

Drawings

FIG. 1 is a schematic diagram showing the design structure of the probe (left) and the primer (right) of the present invention;

FIG. 2 is a schematic diagram showing the working principle of the probe primer of the present invention;

FIG. 3 is a method of designing a probe according to example 1;

FIG. 4 shows a primer design method of example 1;

FIG. 5 shows the effect evaluation of different primer probe combination design methods;

FIG. 6 shows the results of a high sensitivity assay system applied to human SDC2 gene methylation detection;

FIG. 7 shows the results of the detection of novel coronavirus nucleic acids using a high sensitivity system.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be specifically described with reference to the following examples, which are provided for explaining and illustrating the present invention only and are not intended to limit the present invention. Some non-essential modifications and adaptations of the invention according to the foregoing summary will still fall within the scope of the invention.

Example 1

The embodiment provides a design method of a primer and probe structure, which comprises the following steps:

the following first illustrates the design concept of the improved primer and probe structure of the present invention, and the following will be described with reference to the accompanying drawings, to better understand the structure of the primer and probe in the present invention:

as shown in fig. 1 and 2, the technical method of the primer and the probe provided by the invention is as follows:

an improved PCR amplification primer structure, which is q-shaped and 26-44 bases in length, wherein the forward primer comprises 4 parts from the 5 'end to the 3' end:

a first portion of 4-10 bases in length, the sequence of which is a deep sea species sequence, having low homology to human genomic sequences; the preferred base sequence is TAACATA;

the second part has the length of 15-20 bases, the sequence of the second part is complementarily paired with a sequence to be detected of a target gene, and the design principle is followed by the design principle of a fluorescent quantitative detection primer or an Arms-PCR primer;

a third part, the sequence of which is complementary to the first part in reverse direction, and the sequence of which is TATGTTA;

a fourth part of 3-5 bases in length, the sequence of which is complementary paired with a sequence to be detected of the target gene and is separated from the 3' of the second part by 2-3 bases (in the case of ARMS-PCR, the mutation site is arranged in this part);

the first part and the third part of the reverse primer are identical to the forward primer, and the sequences of the second part and the fourth part are respectively complementary matched with the other sequence to be detected of the target gene.

An improved probe structure, which is circular in shape and 28-42 bases in length, comprising five parts from 5 'end to 3':

the first part is 10-15 bases in length, the sequence of the first part is complementarily paired with the sequence to be detected of the target gene, and the design principle of the first part is in accordance with the design principle of a probe;

a second part of 4-6 bases in length and having a sequence of a deep sea species having low homology to the human genome sequence, preferably GGTAGA;

a third part with the length of 10-15 bases, the sequence of which is complementarily paired with the sequence to be detected of the target gene and is separated from the 3' of the first part by 2-3 bases;

a fourth part, the sequence of which is complementary to the second part in reverse direction, the sequence of which is TCTACC;

a fifth part having the same sequence as the third part;

the fluorescent group at the 5 'end of the probe is FAM, VIC, HEX, CY or ROX fluorescent report group suitable for fluorescent quantitative PCR analysis, and the quenching group at the 3' end is TAMRA, BHQ1, BHQ2, MGB or Dabcy1 fluorescent quenching group suitable for fluorescent quantitative PCR.

Example 2

The design method of the primer and the probe of the high-sensitivity fluorescent PCR detection system is examined:

to find a high-sensitivity fluorescent PCR detection system, improvements are made in terms of both primers and probes. The improvement was evaluated by EGFR gene T790M assay system, wherein the PCR template was 0.5% mutant DNA and wild type DNA at a concentration of 10 ng/. Mu.L.

The detection system (I) is shown in the following table 1:

TABLE 1

The detection method is as follows:

95 ℃ for 2min;95 ℃ for 30s,60 ℃ for 30s and 72 ℃ for 30s, and 5 cycles are total; the fluorescence intensity was collected at 95℃for 30s,60℃for 30s, and 72℃for 30s for 40 cycles, and quantitatively analyzed at 60 ℃.

(III) design method of primer and probe:

the design of specific upstream primer, specific downstream primer and specific probe is referred to as follows:

1. the probe design method is as follows:

the probe (P) comprises five parts, wherein the first part is a complementary pairing sequence of a target gene to be detected, the second part is a 6bp deep sea species DNA sequence, the third part is a complementary pairing sequence of the target gene to be detected, the fourth part is a reverse complementary sequence with the second part, and the sequences of the fifth part and the third part are the same (as the D part in figure 3). The specific sequence information is as follows:

P:FAM-5’-ATGCCCTTCGGCTGC--GGTAGA--CTGGACTATGTC--TCTACC--CTGGACTATGTC-3’-TAMRA

2. the primer design method is as follows:

the first part of the specific primer (F/R) is a 7bp deep sea species DNA sequence, the second part is a complementary pairing sequence of a target gene test sequence, the third part is a reverse complementary sequence of the first part, and the fourth part is a complementary pairing sequence of the target gene test sequence (as shown as a part B in figure 4). The specific sequence information of the F/R primer pair is as follows:

F：5’-TAACATA--CTCCACCGTGCAGC--TATGTTA--ATCAT-3’

R：5’-TAACATA--GCAGGTACTGGGA--TATGTTA--CAATA-3’

and fourthly, combining the designed primers and probes, and performing PCR amplification, wherein the obtained PCR amplification result is shown in FIG. 5 (design method 8), and the combination of the primer pair F/R and the probe P is adopted, so that the sensitivity for PCR detection is very high, and 0.5% EGFR gene T790M mutation in 10ng DNA can be well detected.

Comparative example 1

Primer design method contrast: in order to examine the specificity of the primers designed in example 1 of the present invention, the following comparative schemes were set for comparison, respectively:

primer comparison scheme one: the comparative primer 1 (marked as O-F1/O-R1) adopts a conventional ARMS primer design method, and the specific sequence of the O-F1/O-R1 primer pair is as follows:

O-F1:5’-CTCCACCGTGCAGCTCGTTAT-3’

O-R1:5’-GCAGGTACTGGGAGCCAATA-3’

primer comparison scheme II: the first part of the comparison primer 2 (marked as O-F2/O-R2) is a complementary pairing sequence of a target gene sequence to be detected, the second part is a 7bp deep sea species DNA sequence, the third part is in reverse complementary pairing with the second part, and the fourth part is a complementary pairing sequence of the target gene sequence to be detected of the target gene (shown as part A in figure 4). Specific sequence information of the O-F2/O-R2 primer pair is as follows:

O-F2：CTCCACCGTGCAGC--TAACATA--TATGTTA--ATCAT

O-R2：GCAGGTACTGGGA--TAACATA--TATGTTA--CAATA

comparative example 2

The probe design method is compared with that: in order to examine the sensitivity of the probe designed in example 1 of the present invention, the following comparative schemes were set for comparison, respectively:

probe contrast protocol one: the comparison probe 1 (O-P1) adopts a conventional design method, and specific sequence information is as follows:

O-P1:FAM-5’-ATGCCCTTCGGCTGCCTCCTGGACTAT-3’-TAMRA

probe comparison scheme two: the comparison probe 2 (O-P2) comprises three parts, wherein the first part is a 6bp deep sea species DNA sequence, the second part is a complementary pairing sequence with a target gene sequence of a target gene, and the third part is a reverse complementary sequence with the first part (such as part A in figure 3). The specific sequence information is as follows:

O-P2:FAM-5’-GGTAGA--ATGCCCTTCGGCTGCCTCCTGGACTAT--TCTACC-3’-TAMRA

probe comparison scheme three: the comparison probe 3 (O-P3) comprises six parts, wherein the first part is a deep sea species DNA sequence of 5bp, the second part is a complementary pairing sequence with a target gene sequence of a target gene, the third part is a reverse complementary sequence with the first part, the fourth part is a deep sea species DNA sequence of 6bp, the fifth part is a complementary pairing sequence with the target gene sequence of the target gene, and the sixth part is a fourth part reverse complementary sequence (as shown as part B in figure 3). The specific sequence information is as follows:

O-P3:FAM-5’-ATGAA--ATGCCCTTCGGCTGC--TTCAT--GGTAGA--CTGGACTATGTC--TCTACC-3’-TAMRA

probe comparison scheme four: the comparison probe 4 (O-P4) comprises four parts, wherein the first part is a complementary pairing sequence of a target gene sequence to be detected, the second part is a 6bp deep sea species DNA sequence, the third part is a complementary pairing sequence of the target gene sequence to be detected, and the fourth part is a reverse complementary sequence with the second part (such as a part C in figure 3). The specific sequence information is as follows:

O-P4:FAM-5’-ATGCCCTTCGGCTGC--GGTAGA--CTGGACTATGTC--TCTACC-3’-TAMRA

example 3

The specific primer pair and probe combinations obtained in example 2 were compared with the primer pair and probe combinations obtained in comparative examples 1 and 2 for PCR detection of EGFR gene T790M, the detection system was as in example 1, and the methods of combining the primers and probes were as shown in Table 2 below:

TABLE 2

Primer probe combination	Primer pair	Probe with a probe tip
			Design method 1	O-F1/O-R1	O-P1
Design method 2	O-F1/O-R1	O-P2
			Design method 3	O-F1/O-R1	O-P3
Design method 4	O-F1/O-R1	O-P4
			Design method 5	O-F1/O-R1	P
Design method 6	O-F2/O-R2	O-P1
			Design method 7	F/R	O-P1
Design method 8	F/R	P

As shown in FIG. 5, the PCR detection results are shown in FIG. 5, and the primer probe combination (design method 8) of example 2 of the present invention has the best effect on the PCR detection sensitivity of EGFR gene T790M, while the other design methods 1 to 7 of the comparative example have poor PCR detection sensitivity of EGFR gene T790M.

Example 4

The combination of the design method of the primer F/R and the probe P adopted according to the design method 8 in the table 2 is used as a high-sensitivity detection system and applied to the methylation detection of the human SDC2 gene.

The PCR template is simulated stool sample DNA of the intestinal cancer patient with the mutation proportion of 0.1% of 0.5 ng/. Mu.L. Because of the high background of microbial DNA in human fecal samples and low content of human DNA, the detection limit of the existing common detection method can only reach 1 ng/. Mu.L of human fecal sample DNA with a mutation proportion of 1% (such as reference [8] and reference [10 ]).

To examine the effectiveness of the application of the highly sensitive probe and primer system in ARMS-fluorescent PCR, the stool DNA of the intestinal cancer patient with 25% methylation mutation rate of the SDC2 gene was first extracted with a small amount of stool DNA extraction kit (product number: D3141-03) from Guangzhou Mei Biotechnology, and then mixed in a ratio of 0.1% mutation rate to a concentration of 0.5 ng/. Mu.l with the stool DNA of the intestinal cancer patient of ZYMO company, and then with EZ DNA Methylation-Gold from ZYMO ^TM And (3) carrying out Kit (product number: D5005) treatment, designing a conventional specific primer probe system and designing a high-sensitivity specific primer probe system, and evaluating the detection effect of the high-sensitivity specific primer probe system through comparison.

The conventional reaction system is shown in Table 3 below:

TABLE 3 Table 3

The hypersensitive reaction system is shown in Table 4 below:

TABLE 4 Table 4

Real-time fluorescent PCR reactions were performed on an ABI7500 real-time PCR instrument. The reaction conditions are as follows: 95 ℃ for 2min;95 ℃ for 30s,60 ℃ for 30s and 72 ℃ for 30s, and 5 cycles are total; fluorescence signals were collected at 95℃for 30s,60℃for 30s, and 72℃for 30s for 35 cycles.

The specific upstream primer F0 is:

5′-AGGAGGAAGCGAGCGTTTTC-3′

the specific downstream primer R0 is:

5′-AACCCAAAATAAACAAAATCCAC-3′

the specific probe P0 is:

FAM-5′-TCGTAATCGTTGCGGTATTTTGTT-3′-TAMRA

the internal control primer F0-internal is:

5′-TATGGTATTGTTAAGGTTGAGAA-3′

the internal control primer R0-internal is:

5′-CAAAAATAATAACCCTTTTAACT-3′

the internal control probe P0-internal is:

VIC-5′-TTTATTATTATTTTTTAGGAGT-3′-TAMRA

the specific upstream primer F1 is:

5′-TAACATA--AGGAGGAAGCGAGC--TATGTTA--TTTC-3′

the specific downstream primer R1 is:

5′-TAACATA--AACCCAAAATAAACAA--TATGTTA--CCACG-3′

the specific probe P1 is:

FAM-5′-TCGTAATCGT--GGTAGA--CGGTATTTTGTTTC--TCTACC--CGGTATTTTGTTTC-3′-TAMRA

the internal control primer F1-is:

5′-TAACATA--TATGGTATTGTTAAGG--TATGTTA--GAGAA-3′

the internal control primer R1-internal is:

5′-TAACATA--CAAAAATAATAACCCT--TATGTTA--TAACT-3′

the internal control probe P1-is:

VIC-5′-TTTATTATTA--GGTAGA--TTTTAGGAGTGA--TCTACC--TTTTAGGAGTGA-3′-TAMRA

the experimental results are shown in fig. 6, and fig. 6 shows that the amplification of the DNA of the stool sample with the mutation ratio of 0.1% and 0.5 ng/. Mu.L, which is simulated by the amplification of the primer probe system designed by the conventional method, cannot generate a curve, which indicates that the sensitivity is insufficient. And the sensitivity can be obviously improved by amplifying the simulated stool sample DNA with the mutation ratio of 0.1% and 0.5 ng/. Mu.L by a high-sensitivity system.

Example 3

The high sensitivity system detects novel coronavirus nucleic acid.

To examine the effectiveness of the high-sensitivity probe and primer system applied to non-human gene PCR detection, the PCR template is a novel crown with gradient dilutionRhabdoviral ORF1ab and N gene plasmids. The total volume of the reaction system was 20. Mu.l, which included 2. Mu.l of 2X one step u+mix, 0.4. Mu.l of one step u+enzyme mix, 0.25. Mu.M for the upstream and downstream primers, 0.3. Mu.M for the probe, and 1. Mu.l of template. The PCR reactions were performed on an ABI7500 real-time PCR instrument. The reaction conditions are as follows: reverse transcription is carried out for 15min at 55 ℃; followed by a pre-denaturation at 95℃for 30s;95℃for 10s and 60℃for 30s (detection of FAM and VIC fluorescence signals), 45 cycles were performed. Serial 10-fold gradient dilution of standard plasmid DNA, H ₂ O served as a negative control.

The ORF1ab gene upstream primer F2 is:

5′-TAACATA--CGGTGGACAAATTG--TATGTTA--ACCTGT-3′

downstream primer R2

5′-TAACATA--TTCACCTAAATTCAAGGC--TATGTTA--TAAGT-3′

The target sequence of probe P2 is near the upstream primer, probe P2 sequence:

FAM-5′-AAGGAAATTAAGGAGA--GGTAGA--GTTCAGACATT--TCTACC--GTTCAGACATT-3′-TAMRA

the N gene upstream primer F3 is:

5′-TAACAT--AAACACAGTCTGTACCG--TATGTTA--TGCGG-3′

the downstream primer R3 is:

5′-TAACAT--AACGATTGTGCATCAG--TATGTTA--GACTGAA-3′

the target sequence of probe P3 is near the upstream primer, probe P3 sequence:

VIC-5′-TATGTGGAAAGGTTA--GGTAGA--CTGTAGTTGTGA--TCTACC--CTGTAGTTGTGA-3′-TAMRA

the result of detecting the novel coronavirus nucleic acid by the high-sensitivity system is shown in figure 7, and as can be seen from figure 7, the high-sensitivity system provided by the invention has high specificity and sensitivity for detecting the novel coronavirus nucleic acid, and for the novel coronavirus ORF1ab gene and N gene, the detection limit is 10copies/mL, the detection sensitivity is extremely high, and the minimum detection limit of the existing method can only reach 100copies/mL (as in reference [16 ]).

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Claims

1. An improved primer structure for fluorescent PCR detection is characterized by comprising a forward primer and a reverse primer, wherein the forward primer comprises the following four parts from a 5 'end to a 3' end:

a second part: the length is 15-20 bases, and the sequence of the primer is complementary and paired with a sequence to be detected of a target gene;

fourth part: the length is 3-5 bases, the sequence of the primer is complementarily paired with a sequence to be detected of a target gene, and the primer is separated from the second part 3' by 2-3 bases;

2. The improved primer structure of claim 1 wherein the first partial sequence of the forward primer and the reverse primer is TAACATA and the third partial sequence is TATGTTA.

3. The improved primer structure according to claim 1 or 2, wherein the target gene comprises a human SDC2 gene methylation sequence, a novel coronavirus ORF1ab gene, or an N gene.

4. An improved probe structure for high sensitivity fluorescent PCR detection, characterized in that the probe comprises the following five parts from the 5 'end to the 3' end:

a first part: the length is 10-15 bases, and the sequence of the nucleotide sequence is complementary and matched with the sequence to be detected of the target gene;

fifth part: the sequence is identical to the third partial sequence.

5. The improved probe structure of claim 4, wherein the second partial sequence of the probe is ggtag aa and the fourth partial sequence is TCTACC.

6. The improved probe structure according to claim 4 or 5, wherein the target gene comprises a human SDC2 gene methylation sequence, a novel coronavirus ORF1ab gene, or an N gene.

7. The improved probe structure of claim 4 or 5, wherein the 5 'end of the probe is attached with a fluorophore comprising FAM, VIC, HEX, CY or ROX, and the 3' end of the probe is attached with a quenching group comprising TAMRA, BHQ1, BHQ2, MGB or Dabcy1.

8. A high sensitivity fluorescent PCR detection system comprising a primer structure according to any one of claims 1-3 and a probe structure according to any one of claims 4-7.

9. An ARMS-PCR detection method, wherein the primer structure according to any one of claims 1 to 3 and the probe structure according to any one of claims 4 to 7 are used, and a mutation site is provided in the fourth part of the primer.

10. The use of a high sensitivity fluorescent PCR detection system as claimed in claim 8 for trace mutations, ultra low content pathogenic microorganisms and PCR detection of interfering DNA in non-diagnostic tumor fluid biopsies.