CN112725444A

CN112725444A - Primer, probe, kit and detection method for detecting PGR gene expression

Info

Publication number: CN112725444A
Application number: CN202011612877.8A
Authority: CN
Inventors: 杨欢欢; 潘石玄伟; 李璐璐; 梁洪; 郎秋蕾
Original assignee: Hangzhou Lianchuan Gene Diagnosis Technology Co ltd
Current assignee: Hangzhou Lianchuan Gene Diagnosis Technology Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-30

Abstract

The invention discloses a primer pair combination for detecting PGR gene expression, belonging to the technical field of medical molecular biology. The primer pair combination comprises a first primer pair for amplifying PGR genes, and also comprises a second primer pair and a third primer pair for amplifying reference genes ACTB and PGK1 respectively. The invention further discloses a probe combination, which comprises a first probe targeting the amplification product of the first primer pair, and also comprises a second probe targeting the amplification product of the second primer pair and a third probe targeting the amplification product of the third primer pair. The invention also discloses a kit prepared by using the primer pair combination or the combination of the primer pair combination and the probe and a method for detecting the PGR gene. The method for detecting PGR gene expression is not limited in sample type, and has the advantages of more accurate detection result, short detection period, small human error, good repeatability, and more sensitivity and reliability than IHC.

Description

Primer, probe, kit and detection method for detecting PGR gene expression

Technical Field

The invention belongs to the technical field of medical molecular biology, and particularly relates to a primer, a probe, a kit and a detection method for detecting PGR gene expression.

Background

The states of ER alpha, PR, HER-2 and Ki-67 are important indexes for judging molecular typing of early invasive breast cancer (non-specific types), and can be classified into Luminal types (Luminal A and Luminal B), HER-2 overexpression types or Basal-like types (Basal-like) according to different states. Existing studies indicate that the ER α, PR and HER2 status of some advanced breast cancer metastases may change compared to the primary, leading to a consequent change in the strategy for treatment of advanced breast cancer. Statistics shows that the incidence rates of breast cancer cases with positive primary focuses ER, PR and HER2 and negative metastatic focuses are respectively 12% -24%, 22% -41% and 3% -16%, and the incidence rates of breast cancer cases with negative primary focuses and positive metastatic focuses are respectively 5% -9%, 7% -19% and 1% -5%. Due to the heterogeneity of breast cancer tumors, even if the heterogeneity of tumors exists in the same tumor body of early breast cancer, dominant clones may appear in the tumor progression, so that a gene expression profile different from that of the primary tumor appears.

ER α -mediated signaling pathways may lead to down-regulation of HER2 expression, with higher ER α expression levels being less at risk of breast cancer recurrence. PR expression is largely dependent on ER α, with less than 1% probability of expressing PR but not ER α in tumor tissues, decreased expression of ER α and PR in breast cancer recurrence, and a shift in the HR +/HER2+ subtype of breast cancer more readily to the HR-/HER2+ subtype (P ═ 0.006). Studies have shown that chemotherapy and endocrine treatment may be associated with altered era status, which is inconsistent before and after primary and metastatic focus detection, and has a poorer prognosis than patients who are both consistent, especially patients who are positive to negative for era. Recurrent/metastatic breast cancer re-biopsy defines ER α, PR, HER2 status, and is critical for further treatment. The expert advises to re-biopsy the status of ER α, PR and HER2 in all patients with advanced breast cancer where clinically possible and to select a treatment strategy for recurrent/metastatic breast cancer based on the re-biopsy of ER α, PR and HER2 status in combination with clinical and imaging profile considerations. The guidelines suggest that hormone receptor status tests should be performed on all breast invasive and non-invasive cancers.

The current method of genetic status detection is to use Immunohistochemistry (IHC), a semi-quantitative method performed in the pathology department, to detect proteins in tumor tissues using antibodies. However, according to the joint studies of American Society for Clinical Oncology (ASCO) and the American society of pathologists (CAP), limited by the immunohistochemical technique itself, every five breast cancer patients may receive the wrong treatment regimen due to inaccurate IHC detection. Therefore, a high-accuracy gene expression detection method is needed in clinic.

Disclosure of Invention

In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:

the invention provides a primer pair combination for detecting PGR gene expression, which comprises a first primer pair for amplifying the PGR gene, wherein the upstream primer and the downstream primer of the first primer pair respectively have nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.

The PGR gene encodes the Progesterone Receptor (PR). PR is also known as NR3C3, nuclear receptor subunit 3, group C, member 3, a member 3 of the third subfamily of nuclear receptors group C) is an intracellular protein that is activated by the steroid hormone progesterone.

Further, the primer pair combination also comprises a second primer pair and a third primer pair which are respectively used for amplifying reference genes ACTB and PGK1, wherein the upstream primer and the downstream primer of the second primer pair respectively have nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO. 4; the upstream primer and the downstream primer of the third primer pair respectively have nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO. 6.

The ACTB gene is a housekeeping gene, is relatively stable in expression level in most cells, encodes beta-Actin and is one of cytoskeletal Actin. The phosphoglycerate kinase gene (PGK1) encodes phosphoglycerate kinase (PGK). PGK is a key enzyme in glycolysis, is also an essential enzyme for each organism to survive, and belongs to a conserved gene.

A second aspect of the present invention provides a probe set comprising a first probe of an amplification product of the first primer pair according to the first aspect of the present invention, the first probe having a nucleotide sequence shown in SEQ ID NO. 7.

In some embodiments of the invention, the first probe is labeled with a fluorophore. In some embodiments of the invention, the first probe is labeled at the 5 'end with FAM and at the 3' end with BHQ 1.

Further, the probe combination further comprises a second probe targeting an amplification product of the second primer pair according to the first aspect of the invention, and a third probe targeting an amplification product of the third primer pair according to the first aspect of the invention.

In some embodiments of the invention, the second probe and the third probe are labeled with different fluorophores. In some embodiments of the invention, the second probe is labeled at the 5 'end with VIC and at the 3' end with BHQ 2; the 5 'end of the third probe is labeled with CY5, and the 3' end is labeled with BHQ 2.

The third aspect of the present invention provides a kit for detecting PGR gene expression, the kit comprising a primer combination pair according to the first aspect of the present invention and a probe combination according to the second aspect of the present invention.

In some embodiments of the invention, the kit comprises the first primer pair and the first probe. Further, the first probe is labeled with a fluorophore. In some embodiments of the invention, the first probe is labeled at the 5 'end with FAM and at the 3' end with BHQ 1.

In some embodiments of the invention, the kit comprises the first primer pair, the second primer pair, and the third primer pair, and the first probe, the second probe, and the third probe. Further, the second probe and the third probe are labeled with different fluorophores. In some embodiments of the invention, the 5 'end of the first probe is labeled with FAM and the 3' end is labeled with BHQ 1; the 5 'end of the second probe is labeled by VIC, and the 3' end of the second probe is labeled by BHQ 2; the 5 'end of the third probe is labeled with CY5, and the 3' end is labeled with BHQ 2.

In some other embodiments of the invention, the probe may be labeled with other fluorescent and quenching groups, as long as the same detection purpose can be achieved.

Further, the primer pair combination and the probe combination exist in the form of a primer probe mixture. In some embodiments of the invention, the primer probe mixture comprises 0.2-1 μ M of each primer, 0.1-0.2 μ M of each probe, and 3mM MgCl₂And 0.25mM dNTP PCR buffer.

Furthermore, the kit also comprises an enzyme mixed solution, a positive control substance and/or a negative control substance.

In some embodiments of the invention, the enzyme mixture comprises a reverse transcriptase and a DNA polymerase, which can be any commercially available enzyme mixture of a reverse transcriptase and a DNA polymerase. In some embodiments of the invention, the enzyme cocktail comprises 2400U reverse transcriptase and 24-30U DNA polymerase.

In some embodiments of the invention, the positive control is an in vitro transcribed RNA mixture of PGR gene, ACTB gene and PGK1 gene fragment.

In some embodiments of the invention, the negative control is nuclease-free water.

The fourth aspect of the present invention provides a method for detecting PGR gene expression, comprising the steps of:

s1, obtaining a nucleic acid sample of the sample to be detected;

s2, performing RT-qPCR amplification on the nucleic acid sample using the first, second and third primer pairs of the first aspect of the invention, and the first, second and third probes, wherein the first, second and third probes are labeled with different fluorophores;

s3, obtaining Ct values of PGR, reference gene ACTB and PGK1 genes respectively, and obtaining a delta Ct value by using the following formula:

ΔCt＝Ct_PGR-(Ct_ACTB+Ct_PGK1)/2

and (3) judging the PGR gene expression condition according to the delta Ct value: the delta Ct is less than 5 positive, and the PGR gene expression is positive;

the delta Ct is more than or equal to 5 negative, and the PGR gene expression is negative.

In some embodiments of the invention, the first probe is labeled with FAM at the 5 'end and BHQ1 at the 3' end; the 5 'end of the second probe is labeled by VIC, and the 3' end of the second probe is labeled by BHQ 2; the 5 'end of the third probe is labeled with CY5, and the 3' end is labeled with BHQ 2.

In some embodiments of the invention, in step S2, the system for performing amplification RT-qPCR amplification is as follows:

20 μ L system: 19 mu L of primer and probe mixed liquor and 1 mu L of enzyme mixed liquor, wherein,

the primer probe mixed solution is PCR buffer solution containing 0.2-1 MuM of each primer, 0.1-0.2 MuM of each probe, 3mM MgCl2 and 0.25mM dNTP,

the enzyme mixed solution contains 2400U reverse transcriptase and 24-30U DNA polymerase;

the amplification procedure was as follows:

20 minutes at 50 ℃;

3 minutes at 95 ℃;

fluorescence signals were collected at 95 ℃ for 10 seconds, 60 ℃ for 30 seconds, and 45 cycles.

In the present invention, the sample to be tested is a tumor tissue sample, and the nucleic acid sample is an RNA sample.

In some embodiments of the invention, the method detects the PGR gene for non-diagnostic or therapeutic purposes.

Other aspects of the invention also provide:

use of the first primer pair, the second primer pair and the third primer pair, or the first probe, the second probe and the third probe thereof in the preparation of a kit for detecting expression of a PGR gene in a biological sample of a patient according to the first aspect of the invention.

In some embodiments of the invention, the patient is a tumor patient. In some embodiments of the invention, the tumor patient is a breast cancer patient, and accordingly, the sample is a breast tumor sample.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

the invention utilizes the primer probe to carry out RT-qPCR detection on PGR gene expression, and the detection sample type is not limited to paraffin embedded tissues, and fresh tissues and frozen tissues can be detected.

The method for detecting the PGR gene expression has the advantages of small required sample amount, no need of diluting RNA and detection by using a multiple fluorescence PCR technology.

The kit provided by the invention contains an internal control system, so that the sample can be detected more accurately and stably, and the result is ensured to be real and credible.

The detection method has short detection period, and can finish detection within 4 hours. The kit can be used for continuous detection, has simple and objective result interpretation, small human error and good repeatability, and is more sensitive and reliable than IHC. Simple and convenient operation, can realize automation, economy, time saving and batch operation.

Drawings

FIG. 1 shows the results of immunohistochemistry for tumor tissues in example 3 of the present invention.

FIG. 2 shows the amplification curve of the positive control RT-qPCR assay in example 3 of the present invention.

FIG. 3 shows the tumor tissue RNA sample RT-qPCR detection amplification curve in example 3 of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 primers, probes and RT-qPCR kit for detecting PGR gene expression

Specific primers are designed according to the target gene sequence, and the designed primers and probes can be artificially synthesized according to the existing method. The specific primers are as follows:

PGR-Fo：TTACCTGTGGGAGCTGTAAGG(SEQ ID NO.1)

PGR-Re：AGTCATTTCTTCCAGCACATAAG(SEQ ID NO.2)

ACTB-Fo：TTCTACAATGAGCTGCGTGTG(SEQ ID NO.3)

ACTB-Re：GGTGTTGAAGGTCTCAAACATGA(SEQ ID NO.4)

PGK1-Fo：TCCTTAGAGCCAGTTGCTGTAG(SEQ ID NO.5)

PGK1-Re：GCACAGGCTTTCTCCACTT(SEQ ID NO.6)

the probe sequence is as follows:

PGR-Pr：TTGTGCTGCCCTTCCATTGCCCT(SEQ ID NO.7)

the 5 'end was labeled with FAM and the 3' end was labeled with BHQ 1.

ACTB-Pr：CCGTGCTGCTGACCGAGGCC(SEQ ID NO.8)

The 5 'end was labeled with VIC, and the 3' end was labeled with BHQ 2.

PGK1-Pr：TCTCTGCTGGGCAAGGATGTTCTGTTC(SEQ ID NO.9)

Among them, the 5 'end was labeled with CY5, and the 3' end was labeled with BHQ 2.

The RT-qPCR detection kit for detecting PGR gene expression can be prepared by utilizing the primer and the probe, and comprises the following four components:

PGR probe primer mixture: comprises the specific primers (0.2-1 mu M) and the probe (0.1-0.2 mu M) for detecting the expression of the PGR, ACTB and PGK1 genes, PCR buffer solution and 3mM MgCl₂And 0.25mM dNTP.

Enzyme mixture liquid: including 2400U reverse transcriptase and 24-30U DNA polymerase.

Positive control: the RNA mixture of PGR gene, ACTB and PGK1 gene fragment transcribed in vitro.

Negative control: nuclease-free water.

The advantages of using RT-qPCR to detect PGR genes include:

(1) the type of the detected sample is not limited to paraffin embedded tissues, and both fresh tissues and frozen tissues can be detected; the required sample amount is small, the detection is carried out by using a multiple fluorescence PCR technology, and the internal control system is included, so that the sample can be detected more accurately and stably, and the result is ensured to be real and credible;

(2) continuous detection is adopted, the result interpretation is simple and objective, the human error is small, the repeatability is good, and the method is more sensitive and reliable than the IHC

(3) Simple and convenient operation, can realize automation, economy, time saving and batch operation.

Example 2PGR Gene expression RT-qPCR detection method

The PGR gene expression is detected by respectively adopting an immunohistochemical kit and a method, the RT-qPCR kit and the RT-qPCR detection method,

the RT-qPCR detection comprises the following steps:

(1) extracting RNA of a tumor sample to be detected and quantifying the RNA of the sample;

tumor tissues were taken and RNA was extracted according to the instructions of the commercial extraction kit. And quantifying the extracted RNA, wherein the concentration is not lower than 10 ng/mu L, and the sample RNA is directly used as a PCR template.

(2) Preparation of reaction mixture

All components in the kit prepared in example 1 were equilibrated to room temperature and melted, and based on the number of samples to be tested, a reaction mixture was calculated and prepared, and the system preparation table of 20 μ L is shown in table 2.

TABLE 2 preparation of reaction mixture

Adding the reaction mixed solution into an eight-linked PCR reaction tube according to 20 mu L/hole, and adding the RNA of the sample to be detected, the positive control substance and the negative control substance into the corresponding PCR reaction tubes respectively according to 5 mu L/hole for PCR amplification.

(3) PCR amplification

PCR amplification procedure

20 minutes at 50 ℃;

3 minutes at 95 ℃;

95 ℃ for 10 seconds, 60 ℃ for 30 seconds (FAM, VIC, CY5 fluorescence signals were collected), 45 cycles.

By utilizing the steps, the detection period is not more than 4 hours, and the method is convenient and quick.

(4) Analysis of results

Setting a threshold value: and setting a threshold value according to different models. Ct value determination: selecting a positive control reaction tube, and marking a threshold value for the negative control and the sample reaction tube together to obtain Ct values of PGR, reference genes ACTB and PGK1 respectively.

② negative control wells (NTC) should have no amplification curve rising;

and thirdly, each fluorescence signal of the positive reference substance hole (PC) has an amplification curve, and the Ct value is less than 30.

And analyzing the PGR gene detection result by using the delta Ct, and judging the expression condition of the PGR gene in the sample according to the delta Ct value.

ΔCt_Sample(s)＝Ct_{Sample PGR}-Ct_{Reference to the sample}Mean value of

Ct_{Reference to the sample}Average value (Ct)_{Sample ACTB}+Ct_{Sample PGK1})/2

If the delta Ct is less than 5, the detection result is positive, namely the PGR gene expression is positive; if the delta Ct is more than or equal to 5, the detection result is negative, namely the expression of the PGR gene is negative.

Example 3 practical application of RT-qPCR detection kit and RT-qPCR detection method

The RT-qPCR detection kit of the embodiment 1 and the RT-qPCR detection method of the embodiment 2 are adopted to detect the PGR gene expression in the tumor tissue of the breast cancer patient, and the sample is a paraffin-embedded breast cancer tumor tissue sample.

The detection method of immunohistochemistry is as follows:

the Detection of Estrogen Receptor (ER) antigen expressed in breast cancer tumor paraffin-embedded tissue section samples was performed on a vetana BenchMark XT automatic section stainer using a commercial progesterone receptor antibody reagent (immunohistology), No. 2014 No. 3403112, Roche, a vetana Detection kit (ultraView DAB Detection kit, Roche), according to the instructions in the kit:

(1) using slide barcode labels corresponding to the antibody protocol to be implemented;

(2) placing a primary antibody, a corresponding detection kit distributor and auxiliary reagents to be used on a reagent tray, and placing the reagent tray on an automatic section staining instrument;

(3) checking bulk liquid and waste;

(4) placing the slide on an automatic section staining instrument;

(5) starting a staining assay;

(6) after the measurement is completed, the slide is taken down from the automatic section staining instrument;

(7) cleaning with mild detergent or alcohol, and removing the solution of the cover glass;

(8) slides were dehydrated, washed and coverslipped with permanent mounting media in the conventional manner.

As shown in FIG. 1, the results of the detection are positive for ESR expression in the tumor tissue sample as shown in FIG. 1.

The RT-qPCR detection method comprises the following steps:

(1) RNA extraction and sample RNA quantification of tumor sample to be detected

Sample RNA was extracted using a commercial Kit (RNeasy FFPE Kit, 73504, Qiagen) and extracted according to the Kit instructions.

(2) Preparation of reaction mixture

All components in the kit prepared in example 1 were equilibrated to room temperature and melted, and based on the number of samples to be tested, a reaction mixture was calculated and prepared as shown in table 3:

TABLE 3 Table for preparing reaction mixture

Adding the reaction mixed solution into a PCR reaction tube according to 20 mu L/hole, and adding the tumor tissue RNA to be detected, the positive reference substance and the negative reference substance into the corresponding PCR reaction tubes respectively according to 5 mu L/hole for PCR amplification.

(3) PCR amplification

PCR amplification procedure

20 minutes at 50 ℃;

3 minutes at 95 ℃;

(4) Analysis of results

The detection results of the positive control and the tumor tissue sample are shown in fig. 2 and fig. 3, and the specific detection Ct is shown in table 4. Calculated according to the following formula:

ΔCt＝Ct_PGR-(Ct_ACTB+Ct_PGK1)/2

TABLE 4 Ct value measurement results

Thus, the positive control product delta Ct is calculated according to the formula as-0.6605 < 5; sample Δ Ct 0.8135< 5. And judging the sample to be positive in PGR expression according to the delta Ct value, wherein the result is in accordance with the immunohistochemical result.

Example 4 practical application of RT-qPCR detection kit and RT-qPCR detection method

The RT-qPCR detection kit of the embodiment 1 and the RT-qPCR detection method of the embodiment 2 are adopted to detect PGR gene expression in tumor tissues of other breast cancer patients, and the samples are tumor tissue samples.

The RT-qPCR detection method comprises the following steps:

(1) RNA extraction and sample RNA quantification of tumor sample to be detected

(2) Preparation of reaction mixture

All components in the kit prepared in example 1 were equilibrated to room temperature and melted, and based on the number of samples to be tested, a reaction mixture was calculated and prepared as shown in table 5:

TABLE 5 Table for the preparation of the reaction mixture

(3) PCR amplification

PCR amplification procedure

20 minutes at 50 ℃;

3 minutes at 95 ℃;

(4) Analysis of results

The specific detection Ct is shown in Table 5, wherein the Δ Ct value is calculated according to the following formula:

ΔCt＝Ct_PGR-(Ct_ACTB+Ct_PGK1)/2

TABLE 5 results of Ct value detection

And for the sample 5, further repeated detection and verification prove that the PGR gene expression really presents a positive result, which indicates that the detection accuracy of the detection kit and the detection method is higher than that of IHC detection.

The results show that the detection kit and the detection method have very high sensitivity and accuracy for detecting the PGR gene expression level of tumor tissues, particularly breast cancer tumor tissues.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. A primer pair combination for detecting PGR gene expression is characterized by comprising a first primer pair for amplifying PGR gene, wherein the upstream primer and the downstream primer of the first primer pair respectively have nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.

2. The primer pair combination according to claim 1, further comprising a second primer pair and a third primer pair for amplifying reference genes ACTB and PGK1, respectively, wherein upstream and downstream primers of the second primer pair have nucleotide sequences shown as SEQ ID No.3 and SEQ ID No.4, respectively; the upstream primer and the downstream primer of the third primer pair respectively have nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO. 6.

3. A probe set comprising a first probe targeting an amplification product of the first primer pair of claim 1, said first probe having a nucleotide sequence set forth in SEQ ID No. 7.

4. The probe combination of claim 3, wherein the first probe is labeled with a label using fluorescence.

5. The probe combination of claim 3 or 4, further comprising a second probe targeting the amplification product of the second primer pair of claim 2, and a third probe targeting the amplification product of the third primer pair of claim 2.

6. The probe combination of claim 5, wherein the second probe and the third probe are labeled with different fluorescence.

7. A kit for detecting PGR gene expression, comprising the primer set combination of claim 1 and the probe set of claim 3 or 4, or comprising the primer set combination of claim 2 and the probe set of claim 5 or 6.

8. The kit of claim 7, further comprising an enzyme cocktail, a positive control, and/or a negative control.

9. A method for detecting PGR gene expression, comprising the steps of:

s1, obtaining a nucleic acid sample of the sample to be detected;

s2, performing RT-qPCR amplification on the nucleic acid sample by using the primer pair combination of claim 2 and the kit of claim 6;

ΔCt＝Ct_PGR-(Ct_ACTB+Ct_PGK1)/2

and (3) judging the PGR gene expression condition according to the delta Ct value: the delta Ct is less than 5 positive, and the PGR gene expression is positive; the delta Ct is more than or equal to 5 negative, and the PGR gene expression is negative.

10. The method of claim 9, wherein the amplification RT-qPCR amplification is performed in step 2 by the following system:

the primer probe mixture contains 0.2-1 μ M of each primer, 0.1-0.2 μ M of each probe, and 3mM MgCl₂And 0.25mM dNTP in PCR buffer,

the amplification procedure was as follows:

20 minutes at 50 ℃;

3 minutes at 95 ℃;