US 11,753,688 B2
Biomarker for early diagnosis and preoperative assessment of pheochromocytoma/paraganglioma, and application thereof
Jing Liu, Liaoning (CN); Liang Wang, Liaoning (CN); and Jingyun Ma, Liaoning (CN)
Assigned to THE FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIVERSITY, Liaoning (CN)
Appl. No. 17/251,651
Filed by THE FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIVERSITY, Liaoning (CN)
PCT Filed Jun. 25, 2018, PCT No. PCT/CN2018/092624
§ 371(c)(1), (2) Date Dec. 11, 2020,
PCT Pub. No. WO2019/237425, PCT Pub. Date Dec. 19, 2019.
Claims priority of application No. 201810610358.4 (CN), filed on Jun. 14, 2018.
Prior Publication US 2021/0277484 A1, Sep. 9, 2021
Int. Cl. C12Q 1/6886 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 2600/156 (2013.01)] 7 Claims
 
1. A method for detecting in a patient the presence of a exosomal biomarker, comprising:
(a) drawing a peripheral blood sample from a patient having pheochromocytoma or paraganglioma;
(b) centrifuging the sample at 3,000-5,000 rpm to collect serum at 4° C.;
(c) centrifuging the collected serum again at 3,000-5,000 rpm to obtain a supernatant;
(d) centrifuging the supernatant at 100000 g-120000 g, washing and re-suspending the resulting precipitate to obtain an exosome sample;
(e) treating the exosome sample with DNase I;
(f) digesting the treated exosome sample with RNase A to digest external RNA;
(g) chelating the RNase A-digested exosome sample with EDTA;
(h) digesting the exosome sample from step (g) with proteinase K at 50-60° C. for 5-20 min in accordance with the volume ratio of the exosome sample and proteinase K of 1:20-100 to digest the DNase I, RNase A and other external proteins to obtain a nucleic acid sample; and
(i) identifying the nucleic acid sample for the presence or absence of a biomarker, wherein the biomarker contains at least one double-stranded DNA fragment having a variation of SDHB and the variation is the c.281 G>A mutation in the SDHB coding sequence.