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A method for detecting multiple pancreatic cancer tumor markers by multiplex PCR and primers and probes for detection

A tumor marker and pancreatic cancer technology, applied in the field of tumor marker detection, can solve the problems of multiple PCR detection of pancreatic cancer tumor markers, high surgical mortality, low diagnosis rate, etc., and achieve good specificity and sensitivity High, repeatable results

Active Publication Date: 2019-08-27
WEST CHINA HOSPITAL SICHUAN UNIV
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Problems solved by technology

[0002] Pancreatic cancer is a highly deteriorating digestive system tumor with non-specific clinical manifestations. Although medical technology has greatly improved in the past 20 years, there are still many problems in the diagnosis and treatment of pancreatic cancer: the early diagnosis rate is not high, Surgical mortality is high, and the cure rate is very low. The incidence of pancreatic cancer in my country ranks 10th among common tumors, and its fatality rate ranks 4th.
[0006] At present, there is no report on the method of using multiplex PCR to detect multiple pancreatic cancer tumor markers Claudin-4, P53 and 18sRNA

Method used

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  • A method for detecting multiple pancreatic cancer tumor markers by multiplex PCR and primers and probes for detection
  • A method for detecting multiple pancreatic cancer tumor markers by multiplex PCR and primers and probes for detection
  • A method for detecting multiple pancreatic cancer tumor markers by multiplex PCR and primers and probes for detection

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Embodiment 1

[0045] 1. A method for detecting multiple pancreatic cancer tumor markers by multiplex PCR, comprising the following steps:

[0046] (1) According to the detected genes Claudin-4 (NCBI Reference Sequence: NM_001305.4) and P53 (NCBI Reference Sequence: NM_001126112.2), select 18sRNA (NCBI Reference Sequence: NR_003286.2) as a quantitative internal reference, using Beacon Designer 7 software Design primers and probe sequences;

[0047] P53Forward primer: 5'-CAAAAGTCTAGAGCCACC-3' (SEQ ID No: 1);

[0048] P53Reverse primer: 5'-AATGTTTCCTGACTCAGAG-3' (SEQ ID No: 2);

[0049] P53probe: 5'FAM-TCTGACTGCGGCTCCTCC-3'DABCYL (SEQ ID No: 3);

[0050] Claudin-4Forwardprimer: 5'-GCCAGCAACTACGTGTAA-3' (SEQ ID No: 4);

[0051] Claudin-4Reverse primer: 5'-CTCAGTCCAGGGAAGAAC-3' (SEQ ID No: 5);

[0052] Claudin-4probe: 5'HEX-ACGGCTCCACTCTGTTCCTC-3'DABCYL (SEQ ID No: 6);

[0053] 18sRNAForward primer: 5'-GCAGCTAGGAATAATGGA-3'(SEQ ID No:7);

[0054] 18sRNA Reverse primer: 5'-GAATTTCACCTCTAGCG...

experiment example

[0080] According to 2 -△△CT The method was used to quantitatively analyze the relative expression of Claudin-4 in 10 cases of pancreatic cancer tissues, 10 cases of distal normal tissues of pancreatic cancer, and 7 cases of chronic pancreatitis tissues. The specific results are shown in Table 2 and Figure 7 ; The relative expression level of P53, the specific results are shown in Table 3 and Figure 8 .

[0081] Table 2 Relative expression of Claudin-4 in tissue samples

[0082] sample number sample type Relative expression of Claudin-4 1 Normal 0.53687 2 Normal 0.29032 3 Normal 0.50518 4 Normal 0.58400 5 Normal 1.08933 6 Normal 0.09421 7 Normal 0.21821 8 Normal 1.00749 9 Normal 0.53306 10 Normal 0.35515 11 chronic pancreatitis 0.18553 12 chronic pancreatitis 0.25406 13 chronic pancreatitis 0.36738 14 chronic pancreatitis 0.46622 15 chronic pancreatitis 0.61...

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Abstract

The invention provides a method for detecting various pancreatic cancer tumor markers through multiple PCR and primers and probes for detection. The method comprises the following steps that (1) the primers and the probes are designed, specifically, P53 forward and reverse primer sequences and a P53 probe sequence are seen in SEQ ID No:1, 2 and 3 correspondingly, and Claudin-4 forward and reverse primer sequences and a Claudin-4 probe sequence are seen in SEQ ID No: 4, 5 and 6; (2) the pancreatic tissue total RNA is extracted; (3) the RNA concentration and purity are identified; (4) the DNA is removed; and (5) real-time PCR is conducted. By designing the specific primers and probes and optimizing the multiple PCR conditions, the method for detecting the various pancreatic cancer tumor markers through the multiple PCR is established. The method is good in specificity, high in flexibility, reliable in result data, high in repeatability and capable of saving cost and time compared with single detection.

Description

technical field [0001] The invention belongs to the technical field of detection methods of tumor markers, and in particular relates to a method for detecting multiple pancreatic cancer tumor markers by multiplex PCR, primers and probes for detection. Background technique [0002] Pancreatic cancer is a highly deteriorating digestive system tumor with non-specific clinical manifestations. Although medical technology has greatly improved in the past 20 years, there are still many problems in the diagnosis and treatment of pancreatic cancer: the early diagnosis rate is not high, Surgical mortality is high, and the cure rate is very low. The incidence of pancreatic cancer in my country ranks 10th among common tumors, and its fatality rate ranks 4th. Although the serum of patients with pancreatic cancer contains many tumor markers, such as CA19-9 and CA242, as well as carcinoembryonic antigen (CEA), the sensitivity and specificity of serological tumor marker detection are not yet...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12N15/11
Inventor 吴思思陈雪梅丁雨倪银芸朱国念
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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