基于突变扩增系统-实时荧光定量聚合酶链式反应法高灵敏检测甲状腺癌中PTEN<sup>R130*</sup>突变

引用本文: 魏佳, 王瑶琪, 王振新, 孟宪瑛. 基于突变扩增系统-实时荧光定量聚合酶链式反应法高灵敏检测甲状腺癌中PTEN^R130*突变[J]. 分析化学, 2022, 50(5): 701-710. doi: 10.19756/j.issn.0253-3820.221008 shu

Citation: WEI Jia, WANG Yao-Qi, WANG Zhen-Xin, MENG Xian-Ying. Highly Sensitive Detection of PTEN^R130* Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction[J]. Chinese Journal of Analytical Chemistry, 2022, 50(5): 701-710. doi: 10.19756/j.issn.0253-3820.221008 shu

基于突变扩增系统-实时荧光定量聚合酶链式反应法高灵敏检测甲状腺癌中PTEN^R130*突变

魏佳 ^1,2, ,
王瑶琪 ^1, ,
王振新 ^{2,
,} ,
孟宪瑛 ^{1,
,}

1.
吉林大学白求恩第一临床医院甲状腺外科, 长春 130021;
2.
中国科学院长春应用化学研究所, 电分析化学国家重点实验室, 长春 130022

通讯作者: 王振新,E-mail:wangzx@ciac.ac.cn; 孟宪瑛,E-mail:mengxiany@jlu.edu.cn

基金项目:
吉林省科技发展计划项目（No.20210204049YY）和山东省泰山产业领军人才工程项目（No.2019TSCYCX-24）资助。

摘要: 构建了一种基于突变扩增系统-实时荧光定量聚合酶链式反应（Amplification refractory mutation system-realtime fluorescence-quantitative polymerase chain reaction，ARMS-qPCR）的分析方法用于检测甲状腺癌中磷酸酶和张力蛋白同源性缺失（Phosphate and tension homology deleted on chromosome ten，PTEN）基因的PTEN^R130*突变。针对PTEN^R130*突变设计的等位基因特异性和非特异性引物，在两个不同的反应体系中对一份模板同时进行等位基因特异性和非特异性扩增，获得二者Ct值相减得到的ΔCt以实现对等位基因突变频率（Variant allele frequency，VAF）的分析。本研究所构建的ARMS-qPCR法检测线性范围为0.1%~90% VAF，能够检测低至0.01%的VAF，并识别基因组DNA中低至4拷贝的PTEN^R130*突变。使用本方法在24例甲状腺肿瘤样品中成功检出5例VAF＞0.1%的PTEN^R130*突变。此外，ARMS-qPCR法对电化学富集后的PTEN^R130*突变血浆加标样品也表现出良好的检测性能，证明其具有良好的临床实用性和在复杂样本中检测微量PTEN^R130*突变的潜能。

关键词:

English

Highly Sensitive Detection of PTEN^R130* Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction

1.
Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun 130021, China;
2.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

Corresponding author: WANG Zhen-Xin, ; MENG Xian-Ying,

Received Date: 04 January 2022
Revised Date: 18 February 2022
Fund Project:
Supported by the Science and Technology Development Project of Jilin Province, China (No.20210204049YY) and the Shandong Taishan Industry Leading Talent Project, China (No.2019TSCYCX-24).

Abstract: An analytical method based on amplification refractory mutation system-real time fluorescence-quantitative polymerase chain reaction (ARMS-qPCR) was developed for detection of gene mutation PTEN^R130* mutation in thyroid cancer. Allele-specific and reference primers for the PTEN^R130* mutation were designed for allele-specific and reference amplification of each sample at the same time, and the ΔCt value was obtained by Ct-allele-specific value minus Ct-reference value to analyze the variant allele frequency (VAF). The ARMS-qPCR method could detect VAF as low as 0.01%, with a linear range of 0.1% to 90% VAF, and could identify PTEN mutations in genomic DNA as low as 4 copies. This method successfully detected 5 cases of mutation with VAF>0.1% in 24 thyroid tumor samples. In addition, the ARMS-qPCR method also showed good detection performance for PTEN^R130* mutant spiked plasma samples under the electrochemical enrichment, indicating clinical practicability and the potential to detect trace PTEN^R130* mutation in complex samples.

Key words:

1. [1]
  STAMBOLIC V, SUZUKI A, DE LA POMPA J L, BROTHERS G M, MIRTSOS C, SASAKI T, RULAND J, PENNINGER J M, SIDEROVSKI D P, MAK T W. Cell, 1998, 95(1):29-39.STAMBOLIC V, SUZUKI A, DE LA POMPA J L, BROTHERS G M, MIRTSOS C, SASAKI T, RULAND J, PENNINGER J M, SIDEROVSKI D P, MAK T W. Cell, 1998, 95(1):29-39.
2. [2]
  DELGOFFE G M, KOLE T P, ZHENG Y, ZAREK P E, MATTHEWS K L, XIAO B, WORLEY P F, KOZMA S C, POWELL J D. Immunity, 2009, 30(6):832-844.DELGOFFE G M, KOLE T P, ZHENG Y, ZAREK P E, MATTHEWS K L, XIAO B, WORLEY P F, KOZMA S C, POWELL J D. Immunity, 2009, 30(6):832-844.
3. [3]
  CALDERON L, SCHINDLER K, MALIN S G, SCHEBESTA A, SUN Q, SCHWICKERT T, ALBERTI C, FISCHER M, JARITZ M, TAGOH H, EBERT A, MINNICH M, LISTON A, COCHELLA L, BUSSLINGER M. Sci. Immunol., 2021, 6(61):e5003.CALDERON L, SCHINDLER K, MALIN S G, SCHEBESTA A, SUN Q, SCHWICKERT T, ALBERTI C, FISCHER M, JARITZ M, TAGOH H, EBERT A, MINNICH M, LISTON A, COCHELLA L, BUSSLINGER M. Sci. Immunol., 2021, 6(61):e5003.
4. [4]
  YANG M, SCHIAPPARELLI P, NGUYEN H N, IGARASHI A, ZHANG Q, ABBADI S, AMZEL L M, SESAKI H, QUINONES-HINOJOSA A, IIJIMA M. Oncogene, 2017, 36(26):3673-3685.YANG M, SCHIAPPARELLI P, NGUYEN H N, IGARASHI A, ZHANG Q, ABBADI S, AMZEL L M, SESAKI H, QUINONES-HINOJOSA A, IIJIMA M. Oncogene, 2017, 36(26):3673-3685.
5. [5]
  XU W, ZHANG M J, LI Y, WANG Y, WANG K, CHEN Q Y, ZHANG R J, SONG W W, HUANG Q Q, ZHAO W H, WU J Q. Cancer Cell Int., 2021, 21(1):30.XU W, ZHANG M J, LI Y, WANG Y, WANG K, CHEN Q Y, ZHANG R J, SONG W W, HUANG Q Q, ZHAO W H, WU J Q. Cancer Cell Int., 2021, 21(1):30.
6. [6]
  LI L H, YUE P L, SONG Q Q, YEN T T, ASAKA S, WANG T L, BEAVIS A L, FADER A N, JIAO Y C, YUAN G W, SHIH I M, SONG Y. J. Pathol., 2021, 253(1):119-128.LI L H, YUE P L, SONG Q Q, YEN T T, ASAKA S, WANG T L, BEAVIS A L, FADER A N, JIAO Y C, YUAN G W, SHIH I M, SONG Y. J. Pathol., 2021, 253(1):119-128.
7. [7]
  FAGIN J A, WELLS S A. N. Engl. J. Med., 2016, 375(11):1054-1067.FAGIN J A, WELLS S A. N. Engl. J. Med., 2016, 375(11):1054-1067.
8. [8]
  DAHIA P L, MARSH D J, ZHENG Z, ZEDENIUS J, KOMMINOTH P, FRISK T, WALLIN G, PARSONS R, LONGY M, LARSSON C, ENG C. Cancer Res., 1997, 57(21):4710-4713.DAHIA P L, MARSH D J, ZHENG Z, ZEDENIUS J, KOMMINOTH P, FRISK T, WALLIN G, PARSONS R, LONGY M, LARSSON C, ENG C. Cancer Res., 1997, 57(21):4710-4713.
9. [9]
  LANDA I, POZDEYEV N, KORCH C, MARLOW L A, SMALLRIDGE R C, COPLAND J A, HENDERSON Y C, LAI S Y, CLAYMAN G L, ONODA N, TAN A C, GARCIA-RENDUELES M E R, KNAUF J A, HAUGEN B R, FAGIN J A, SCHWEPPE R E. Clin. Cancer Res., 2019, 25(10):3141-3151.LANDA I, POZDEYEV N, KORCH C, MARLOW L A, SMALLRIDGE R C, COPLAND J A, HENDERSON Y C, LAI S Y, CLAYMAN G L, ONODA N, TAN A C, GARCIA-RENDUELES M E R, KNAUF J A, HAUGEN B R, FAGIN J A, SCHWEPPE R E. Clin. Cancer Res., 2019, 25(10):3141-3151.
10. [10]
  DAVIES L, HOANG J K. Lancet Diabetes Endocrinol., 2021, 9(1):11-12.DAVIES L, HOANG J K. Lancet Diabetes Endocrinol., 2021, 9(1):11-12.
11. [11]
  MIRANDA-FILHO A, LORTET-TIEULENT J, BRAY F, CAO B, FRANCESCHI S, VACCARELLA S, DAL MASO L. Lancet Diabetes Endocrinol., 2021, 9(4):225-234.MIRANDA-FILHO A, LORTET-TIEULENT J, BRAY F, CAO B, FRANCESCHI S, VACCARELLA S, DAL MASO L. Lancet Diabetes Endocrinol., 2021, 9(4):225-234.
12. [12]
  FAN D, MA J, BELL A C, GROEN A H, OLSEN K S, LOK B H, LEEMAN J E, ANDERSON E, RIAZ N, MCBRIDE S, GANLY I, SHAHA A R, SHERMAN E J, TSAI C J, KANG J J, LEE N Y. Cancer, 2020, 126(2):444-452.FAN D, MA J, BELL A C, GROEN A H, OLSEN K S, LOK B H, LEEMAN J E, ANDERSON E, RIAZ N, MCBRIDE S, GANLY I, SHAHA A R, SHERMAN E J, TSAI C J, KANG J J, LEE N Y. Cancer, 2020, 126(2):444-452.
13. [13]
  DURANTE C, GRANI G, LAMARTINA L, FILETTI S, MANDEL S J, COOPER D S. JAMA, J. Am. Med. Assoc., 2018, 319(9):914-924.DURANTE C, GRANI G, LAMARTINA L, FILETTI S, MANDEL S J, COOPER D S. JAMA, J. Am. Med. Assoc., 2018, 319(9):914-924.
14. [14]
  LIU M, RUAN M, CHEN L. Med. Oncol., 2014, 31(6):973.LIU M, RUAN M, CHEN L. Med. Oncol., 2014, 31(6):973.
15. [15]
  CHEN Huan, HAN Xin-Tao, WEI Jia, LIU Jia-Nan, WANG Guo-Qing, LIU Wan-Jian, TIAN Yong-Shuai, MENG Xian-Ying, WANG Zhen-Xin. Chin. J. Anal. Chem., 2021, 49(6):893-906. 陈欢, 韩鑫涛, 魏佳, 刘嘉男, 王国庆, 刘万建, 田永帅, 孟宪瑛, 王振新. 分析化学, 2021, 49(6):893-906.
16. [16]
  HUBER F, LANG H P, GLATZ K, RIMOLDI D, MEYER E, GERBER C. Nano Lett., 2016, 16(9):5373-5377.HUBER F, LANG H P, GLATZ K, RIMOLDI D, MEYER E, GERBER C. Nano Lett., 2016, 16(9):5373-5377.
17. [17]
  LI P, HE H, WANG Z, FENG M, JIN H, WU Y, ZHANG L, ZHANG L, TANG X. Anal. Chem., 2016, 88(1):883-889.LI P, HE H, WANG Z, FENG M, JIN H, WU Y, ZHANG L, ZHANG L, TANG X. Anal. Chem., 2016, 88(1):883-889.
18. [18]
  TU M, CHIA D, WEI F, WONG D. Analyst, 2016, 141(2):393-402.TU M, CHIA D, WEI F, WONG D. Analyst, 2016, 141(2):393-402.
19. [19]
  ZAPPE K, PIRKER C, MIEDL H, SCHREIBER M, HEFFETER P, PFEILER G, HACKER S, HASLIK W, SPIEGL-KREINECKER S, CICHNA-MARKL M. Int. J. Mol. Sci., 2021, 22(22):12527.ZAPPE K, PIRKER C, MIEDL H, SCHREIBER M, HEFFETER P, PFEILER G, HACKER S, HASLIK W, SPIEGL-KREINECKER S, CICHNA-MARKL M. Int. J. Mol. Sci., 2021, 22(22):12527.
20. [20]
  DU Y, POTHUKUCHY A, GOLLIHAR J D, NOURANI A, LI B, ELLINGTON A D. Angew. Chem., Int. Ed., 2017, 56(4):992-996DU Y, POTHUKUCHY A, GOLLIHAR J D, NOURANI A, LI B, ELLINGTON A D. Angew. Chem., Int. Ed., 2017, 56(4):992-996
21. [21]
  WANG Y, TIAN K, SHI R, GU A, PENNELLA M, ALBERTS L, GATES K S, LI G, FAN H, WANG M X, GU L Q. ACS Sens., 2017, 2(7):975-981WANG Y, TIAN K, SHI R, GU A, PENNELLA M, ALBERTS L, GATES K S, LI G, FAN H, WANG M X, GU L Q. ACS Sens., 2017, 2(7):975-981
22. [22]
  CHEN D, QI W, ZHANG P, ZHANG Y, LIU Y, GUAN H, WANG L. Pathology, 2018, 214(2):303-307CHEN D, QI W, ZHANG P, ZHANG Y, LIU Y, GUAN H, WANG L. Pathology, 2018, 214(2):303-307
23. [23]
  MUKHERJEE S, SATHANOORI M, MA Z, ANDREATTA M, LENNON P A, WHEELER S R, PRESCOTT J L, COLDREN C, CASEY T, RIETZ H, FASIG K, WOODFORD R, HARTLEY T, SPENCE D, DONNELAN W, BERDEJA J, FLINN I, KOZYR N, BOUZYK M, CORRELL M, HO H, KRAVTSOV V, TUNNEL D, CHANDRA P. Canc. Genet., 2017, 216:128-141MUKHERJEE S, SATHANOORI M, MA Z, ANDREATTA M, LENNON P A, WHEELER S R, PRESCOTT J L, COLDREN C, CASEY T, RIETZ H, FASIG K, WOODFORD R, HARTLEY T, SPENCE D, DONNELAN W, BERDEJA J, FLINN I, KOZYR N, BOUZYK M, CORRELL M, HO H, KRAVTSOV V, TUNNEL D, CHANDRA P. Canc. Genet., 2017, 216:128-141
24. [24]
  EMAUS M N, ANDERSON J L. Anal. Chim. Acta, 2020, 1124:184-193.EMAUS M N, ANDERSON J L. Anal. Chim. Acta, 2020, 1124:184-193.
25. [25]
  BOTEZATU I V, KONDRATOVA V N, SHELEPOV V P, MAZURENKO N N, TSYGANOVA I V, SUSOVA O Y, LICHTENSTEIN A V. Anal. Biochem., 2020, 590:113517.BOTEZATU I V, KONDRATOVA V N, SHELEPOV V P, MAZURENKO N N, TSYGANOVA I V, SUSOVA O Y, LICHTENSTEIN A V. Anal. Biochem., 2020, 590:113517.
26. [26]
  NAKAMURA Y, TANIGUCHI H, IKEDA M, BANDO H, KATO K, MORIZANE C, ESAKI T, KOMATSU Y, KAWAMOTO Y, TAKAHASHI N, UENO M, KAGAWA Y, NISHINA T, KATO T, YAMAMOTO Y, FURUSE J, DENDA T, KAWAKAMI H, OKI E, NAKAJIMA T, NISHIDA N, YAMAGUCHI K, YASUI H, GOTO M, MATSUHASHI N, OHTSUBO K, YAMAZAKI K, TSUJI A, OKAMOTO W, TSUCHIHARA K, YAMANAKA T, MIKI I, SAKAMOTO Y, ICHIKI H, HATA M, YAMASHITA R, OHTSU A, ODEGAARD J I, YOSHINO T. Nat. Med., 2020, 26(12):1859-1864.NAKAMURA Y, TANIGUCHI H, IKEDA M, BANDO H, KATO K, MORIZANE C, ESAKI T, KOMATSU Y, KAWAMOTO Y, TAKAHASHI N, UENO M, KAGAWA Y, NISHINA T, KATO T, YAMAMOTO Y, FURUSE J, DENDA T, KAWAKAMI H, OKI E, NAKAJIMA T, NISHIDA N, YAMAGUCHI K, YASUI H, GOTO M, MATSUHASHI N, OHTSUBO K, YAMAZAKI K, TSUJI A, OKAMOTO W, TSUCHIHARA K, YAMANAKA T, MIKI I, SAKAMOTO Y, ICHIKI H, HATA M, YAMASHITA R, OHTSU A, ODEGAARD J I, YOSHINO T. Nat. Med., 2020, 26(12):1859-1864.
27. [27]
  LEBOFSKY R, DECRAENE C, BERNARD V, KAMAL M, BLIN A, LEROY Q, RIO FRIO T, PIERRON G, CALLENS C, BIECHE I, SALIOU A, MADIC J, ROULEAU E, BIDARD F C, LANTZ O, STERN M H, LE TOURNEAU C, PIERGA J Y. Mol. Oncol., 2015, 9(4):783-790.LEBOFSKY R, DECRAENE C, BERNARD V, KAMAL M, BLIN A, LEROY Q, RIO FRIO T, PIERRON G, CALLENS C, BIECHE I, SALIOU A, MADIC J, ROULEAU E, BIDARD F C, LANTZ O, STERN M H, LE TOURNEAU C, PIERGA J Y. Mol. Oncol., 2015, 9(4):783-790.
28. [28]
  NEWTON C R, GRAHAM A, HEPTINSTALL L E, POWELL S J, SUMMERS C, KALSHEKER N, SMITH J C, MARKHAM A F. Nucleic Acids Res., 1989, 17(7):2503-2516.NEWTON C R, GRAHAM A, HEPTINSTALL L E, POWELL S J, SUMMERS C, KALSHEKER N, SMITH J C, MARKHAM A F. Nucleic Acids Res., 1989, 17(7):2503-2516.
29. [29]
  YU P C, TAN L C, ZHU X L, SHI X, CHERNIKOV R, SEMENOV A, ZHANG L, MA B, WANG Y, ZHOU X Y, JI Q H, WEI W J, WANG Y L. Endocr. Pract., 2021, 27(7):698-705.YU P C, TAN L C, ZHU X L, SHI X, CHERNIKOV R, SEMENOV A, ZHANG L, MA B, WANG Y, ZHOU X Y, JI Q H, WEI W J, WANG Y L. Endocr. Pract., 2021, 27(7):698-705.
30. [30]
  XU S, DUAN Y, LOU L, TANG F, SHOU J, WANG G. Oncol. Lett., 2016, 12(5):4238-4244.XU S, DUAN Y, LOU L, TANG F, SHOU J, WANG G. Oncol. Lett., 2016, 12(5):4238-4244.
31. [31]
  WEI J, ZHAO Z, GAO J X, WANG Y Q, MA L N, MENG X Y, WANG Z X. ACS Omega, 2020, 5(10):5365-5371.WEI J, ZHAO Z, GAO J X, WANG Y Q, MA L N, MENG X Y, WANG Z X. ACS Omega, 2020, 5(10):5365-5371.
32. [32]
  KOMIJANI M, SHAHIN K, AZHAR E I, BAHRAM M. Methods Mol. Biol., 2022, 2392:93-99.KOMIJANI M, SHAHIN K, AZHAR E I, BAHRAM M. Methods Mol. Biol., 2022, 2392:93-99.
33. [33]
  BRADLEY K M, ELMORE J B, BREYER J P, YASPAN B L, JESSEN J R, KNAPIK E W, SMITH J R. Genome Biol., 2007, 8(4):R55.BRADLEY K M, ELMORE J B, BREYER J P, YASPAN B L, JESSEN J R, KNAPIK E W, SMITH J R. Genome Biol., 2007, 8(4):R55.
34. [34]
  BUTCHER L M, MEABURN E, LIU L, FERNANDES C, HILL L, AL-CHALABI A, PLOMIN R, SCHALKWYK L, CRAIG I W. Behav. Genet., 2004, 34(5):549-555.BUTCHER L M, MEABURN E, LIU L, FERNANDES C, HILL L, AL-CHALABI A, PLOMIN R, SCHALKWYK L, CRAIG I W. Behav. Genet., 2004, 34(5):549-555.

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沈阳化工大学材料科学与工程学院沈阳 110142

基于突变扩增系统-实时荧光定量聚合酶链式反应法高灵敏检测甲状腺癌中PTEN^R130*突变

通讯作者: 王振新,E-mail:wangzx@ciac.ac.cn; 孟宪瑛,E-mail:mengxiany@jlu.edu.cn

English

Highly Sensitive Detection of PTEN^R130* Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction

Corresponding author: WANG Zhen-Xin, ; MENG Xian-Ying,

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

基于突变扩增系统-实时荧光定量聚合酶链式反应法高灵敏检测甲状腺癌中PTENR130*突变

通讯作者: 王振新,E-mail:wangzx@ciac.ac.cn; 孟宪瑛,E-mail:mengxiany@jlu.edu.cn

English

Highly Sensitive Detection of PTENR130* Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction

Corresponding author: WANG Zhen-Xin, ; MENG Xian-Ying,

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

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Highly Sensitive Detection of PTEN^R130* Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction

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