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Kit for analyzing HER2 gene copy number variation by combining multiple internal references with sequential probability ratio test and use method

A technology of gene copy number and sequential probability ratio, which is applied in the field of biomedical nucleic acid detection, can solve the problem of low accuracy of HER2 gene copy number variation status, and achieve the goal of avoiding subjective errors, reducing the number of samples, and improving accuracy Effect

Active Publication Date: 2020-12-08
北京爱普拜生物技术有限公司
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AI Technical Summary

Problems solved by technology

[0004] The present invention aims to provide a kit for analyzing HER2 gene copy number variation with multiple internal references combined with SPRT and a method of use to solve the technical problem of low accuracy of IHC / FISH detection of HER2 gene copy number variation

Method used

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  • Kit for analyzing HER2 gene copy number variation by combining multiple internal references with sequential probability ratio test and use method
  • Kit for analyzing HER2 gene copy number variation by combining multiple internal references with sequential probability ratio test and use method
  • Kit for analyzing HER2 gene copy number variation by combining multiple internal references with sequential probability ratio test and use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Establishment of a kit for analyzing HER2 gene copy number variation with multiple internal references combined with SPRT and its application method

[0024] In this example, a method for detecting HER2 gene copy number variation was established using paraffin samples of breast cancer cell lines, and the detection ability of the method was preliminarily evaluated. Embodiments 1 to 3 are all according to figure 1 The flow chart shown is for the use of the kit and the statistical analysis of subsequent data.

[0025] 1. The nucleotide sequences of the primers and probe nuclei involved in the present invention are shown in Table 1.

[0026] Table 1, the nucleotide sequence list of primers and probes

[0027] serial number name Nucleotide sequence SEQ ID NO: 1 HER2-FPrimer 5'-CTAGCACCTTGCTAAGCA-3' SEQ ID NO: 2 HER2-RPrimer 5'-GAGCACCATTCACAGAAA-3' SEQ ID NO: 3 CEP17-FPrimer 5'-TCTGCCTAATCTACCAATG-3' SEQ ID NO: 4 CE...

Embodiment 2

[0057] Example 2 The state of HER2 gene copy number variation in 10 cases of paraffin tissue section samples was detected using the kit and method of use described in the present invention, and compared with the accuracy of the ratio directly calculated using the CEP17 single internal reference, CEP17 / EIF2C1 The calculated confidence interval and HER2 / CEP17 threshold line are the same as those in Example 1.

[0058] 1. Sample preparation: 10 paraffin samples of breast cancer cell lines.

[0059] 2. The procedures for DNA extraction and digital PCR detection are the same as in Example 1.

[0060] 3. The results of digital PCR detection and SPRT analysis are shown in Table 7 and Table 8.

[0061] Table 7. Detection results of HER2 gene copy number variation status in paraffin samples of 10 breast cancer cell lines using the kit and method of use according to the present invention

[0062]

[0063] Table 8. SPRT analysis results of each sample in 10 breast cancer cell line p...

Embodiment 3

[0068] Example 3 Comparison of the kit and method of use of the present invention with the CEP17 single internal reference analysis method

[0069] 1. Combining the two parts of sample information involved in Example 1 and Example 2, see Table 10 for the comparison results of the kit and usage method of the present invention and the CEP17 single internal reference analysis method.

[0070] Table 10. In 40 cases of breast cancer cell line paraffin samples, the comparison between using the kit and using method of the present invention and CEP17 single internal reference analysis method

[0071]

[0072] 2. After a comprehensive analysis, it was found that in 40 samples, using the CEP17 single internal reference analysis method to detect the status of HER2 gene copy number variation showed that the detection results of samples 26, 34, 35, 37, and 40 were consistent with IHC / FISH results. Conformity, the accuracy rate is 87.5% (35 / 40, 95% CI 73.89%-94.54%), and the status of ch...

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Abstract

The invention relates to a kit and a method for analyzing HER2 gene copy number variation based on a digital PCR method and by combining multiple internal references with sequential probability ratiotest (SPRT). The kit comprises a digital PCR enzyme premixed solution, a primer probe mixed solution for detecting a target gene HER2 and internal reference CEP17 and EIF2C1, a fluorescein sodium saltsolution and ddH2O. By comparing the expression levels of the two reference genes, whether the 17 # chromosome where the HER2 gene is located has the conditions of polysome, monomer, centromere amplification and the like or not can be accurately and effectively judged. Besides, accurate interpretation of the HER2 gene copy number variation state can be completed through SPRT analysis, the accuracy of detection result judgment is remarkably improved, and the kit and the method can be used as a kit and a method for HER2 gene copy number variation primary detection and have the irreplaceable advantages of other methods.

Description

technical field [0001] The invention relates to a kit for analyzing HER2 gene copy number variation with multiple internal references combined with a sequential probability ratio test and a method for using it, belonging to the technical field of biomedical nucleic acid detection. Background technique [0002] Human epidermal growth factor receptor 2 (HER2) is a proto-oncogene encoding tyrosine kinase activity, located on chromosome 17. The detection of HER2 gene copy number variation is the basis for judging the prognosis of breast cancer and formulating effective treatment plans (including reference indicators for hormone therapy and chemotherapy), and is also a prerequisite for whether targeted drug therapy can be adopted. At present, the detection methods of HER2 gene copy number variation widely carried out in hospitals are mainly immunohistochemistry (IHC) method and fluorescence in situ hybridization (FISH) method. Clinical data show that the IHC method cannot accura...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11G16B30/00
CPCC12Q1/686C12Q1/6886C12Q2600/118C12Q2600/156C12Q2600/166G16B30/00C12Q2537/16C12Q2537/165C12Q2563/159
Inventor 龚建于祥春高敏冯晓燕林挺
Owner 北京爱普拜生物技术有限公司
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