Integrative analytical and statistical framework for optimization of multiplex qPCR detection of <italic style="font-style: italic">TGFBI</italic> mutations in refractive surgery candidates

Yunfeng GU; Liping MAO; Xiaoling LI; Kangxuan SUN; Qiuruo JIANG; Wenhui WU; Yangyang SHEN; Shihao CHEN; Meiqin ZHENG; Yi XU

doi:10.1631/jzus.B2500747

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Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates

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Research Article | Updated：2026-06-23

Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates
Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates
屈光手术患者TGFBI突变多重qPCR检测体系的建立
Journal of Zhejiang University-SCIENCE B Vol. 27, Issue 6, Pages: 645-655(2026)
Affiliations：

1.National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China
2.Institute of PSI Genomics, Wenzhou Global Eye & Vision Innovation Center, Wenzhou 325024, China
3.Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China
Funds：
DOI：10.1631/jzus.B2500747
CLC：
CSTR：
Received：19 November 2025，

Revised：2026-03-24，

Published：15 June 2026
Accepted：

顾云峰,毛丽萍,李小玲等.屈光手术患者,TGFBI突变多重qPCR检测体系的建立[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(06):645-655.

Yunfeng GU, Liping MAO, Xiaoling LI, et al. Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(6): 645-655. DOI: 10.1631/jzus.B2500747.
顾云峰,毛丽萍,李小玲等.屈光手术患者,TGFBI突变多重qPCR检测体系的建立[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(06):645-655. DOI： 10.1631/jzus.B2500747.

Yunfeng GU, Liping MAO, Xiaoling LI, et al. Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(6): 645-655. DOI: 10.1631/jzus.B2500747. DOI：

摘要

TGFBI

基因突变是导致角膜营养不良的重要病因，也是屈光手术术前必须排查的遗传风险因素。然而，目前仍缺乏稳定、灵敏且可标准化的多重检测体系。本研究构建了一套整合分析与统计框架，用于优化针对

TGFBI

高频突变位点的多重等位基因特异性定量聚合酶链反应（qPCR）检测方法，并系统评估其临床应用价值。通过条件优化、阈值确定、重复性与特异性验证，建立了可同时检测R124C/L/H、R555W/Q等关键突变的多重qPCR体系。统计分析结果表明，该方法扩增效率良好、重复性高且检出限低，在梯度稀释样本中表现出稳定的定量能力，且无明显非特异性扩增。受试者工作特征曲线（ROC）与概率单位（Probit）分析进一步验证了体系的准确性与可靠性，能够有效检出微量模板中突变序列。将该体系应用于大规模屈光手术候选者术前筛查，成功检出了低频

TGFBI

突变携带者，且结果与Sanger测序完全一致。上述结果证实，基于整合分析与统计优化的多重qPCR体系，能够高效、可靠地用于

TGFBI

突变筛查，为屈光手术提供标准化的术前遗传风险评估工具，也为其他遗传病位点的多重检测优化提供了可借鉴的统计与方法学框架。

Abstract

Refractive surgery can unmask or accelerate transforming growth factor-β-induced (

TGFBI

)-related corneal dystrophies that are undetectable by routine slit-lamp examination

creating a clear need for a rapid

standardized

preoperative genetic screening. We developed a multiplex

allele-specific real-time quantitative polymerase chain reaction (qPCR) panel

targeting five high-frequency

TGFBI

hotspots (R124C/L/H

R555W/Q) and built a statistics-driven analytical framework to optimize assay decisions. Receiver operating characteristic (ROC) analysis defined locus-specific cycle threshold (

) cut-offs that were harmonized to a single decision threshold (

=36) to simplify deployment. Analytical sensitivity was established by Probit modeling of serial two-fold dilutions

and confirmed by ≥20 replicates per level. In a 158-sample validation set (38 mutation-positive; 120 negative)

qPCR agreed perfectly with Sanger sequencing (Cohen’s kappa coefficient (

)=1.0). Probit analysis yielded locus-specific limit of detection (LoD) values ranging from 0.035 to 0.200 ng/µL; at 0.200 ng/µL

the detection rate was over 95%. Repeatability and intermediate precision were high (

coefficient of variation (CV) 0.34%–1.21%). No cross-reactivity was observed against non-target

TGFBI

variants or other ophthalmic genes

and interference from blood

oral flora/rinse

or toothpaste produced small

bounded shifts (approximately -7.8% to +2.8%). Calibration with serial dilutions demonstrated linear

–log(copy) relationships suitable for routine quality control. Prospective screening of 10 055 refractive surgery candidates identified six

TGFBI

carriers (0.06%) harboring R124H (including one homozygote)

R124L

R124C

or R555W mutation

all confirmed by Sanger sequencing. This study established a clinically applicable

statistically optimized multiplex qPCR platform that integrated ROC-derived cut-offs and Probit-defined LoD with rigorous evaluations of precision

specificity

and robustness

enabling large-scale population implementation. Positive screening results guide clinical decision-making through a standardized post-screening workflow

and the targeted hotspot screening strategy serves as a cost-effective

first-tier high-throughput approach for preoperative risk assessment. The framework provides a transparent

reproducible path to standardize preoperative

TGFBI

screening and reduce iatrogenic risk in refractive surgery candidates.

关键词

Keywords

references

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Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates

Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates

DOI：10.1631/jzus.B2500747

摘要

Abstract

关键词

Keywords

references