柑橘黄龙病菌双重实时荧光PCR检测方法的建立

doi:10.16420/j.issn.0513-353x.2021-0502

摘要/Abstract

摘要：

建立基于双重荧光定量PCR检测柑橘黄龙病菌（Candidatus Liberibacter asiaticus，CLas）的方法，并对其准确性进行验证。通过受试者工作特征（ROC）曲线、净重分类改善指数（NRI）和综合判别改善指数（IDI）分析7对CLas实时荧光PCR引物及其联合检测的准确性，评价RNRf/RNRr联合CLas-4G/HLBr的双重实时荧光PCR对CLas的检测价值。ROC曲线分析结果显示，表现最好的引物是RNRf/RNRr，其曲线下面积（AUC）为0.971，高于其他6对引物；其次是CLas-4G/HLBr引物，AUC为0.966；在双引物联合检测中，RNRf/RNRr + CLas-4G/HLBr引物串联检测、CQULA04F/CQULA04R + RNRf/RNRr引物并联检测的相关性能指标较优，AUC分别为0.963和0.966，并且RNRf/RNRr + CLas-4G/HLBr引物串联检测同时具备较高的敏感性（85.19%）和特异性（99.25%）。NRI分析结果表明RNRf/RNRr + CLas-4G/HLBr串联检测的诊断能力相较于LJ900f/LJ900r的单一引物实时荧光PCR显著提升了6.00%（P < 0.05）。使用RNRf/RNRr和CLas-4G/HLBr两对引物进行双重实时荧光PCR，发现RNRf/RNRr + CLas-4G/HLBr双重实时荧光PCR比单一引物实时荧光PCR有更高的灵敏度。因此，RNRf/RNRr + CLas-4G/HLBr双重实时荧光PCR可作为柑橘黄龙病低发区的早期诊断工具。

关键词: 柑橘黄龙病, 实时荧光定量PCR, 引物, 联合诊断

Abstract:

This study aimed to establish a duplex real-time PCR assay to detect‘Candidatus Liberibacter asiaticus’（CLas）. Receiver operating characteristic（ROC）curve，net reclassification improvement（NRI），and integrated discrimination improvement（IDI）were used to analyze the accuracy of seven real-time PCR primer sets and their pair-wise combination detections，and evaluate the diagnostic value of duplex real-time PCR with primer sets RNRf/RNRr and CLas-4G/HLBr. The best diagnostic performance in the single primer assay was RNRf/RNRr with an area under the curve（AUC）of 0.971，followed by CLas-4G/HLBr with an AUC of 0.966. In combined real-time PCR detection by two primer sets，both the tandem use of primer set RNRf/RNRr + CLas-4G/HLBr and the parallel use of primer set CQULA04F/CQULA04R + RNRf/RNRr showed good diagnostic performances with AUCs of 0.963 and 0.966，and the RNRf/RNRr + CLas-4G/HLBr tandem assay had both high（85.19%）and specificity （99.25%）. NRI analysis showed that the predictive ability of the tandem use of primer set RNRf/RNRr + CLas-4G/HLBr was significantly increased by 6.00% compared with the single use of primer set LJ900f/LJ900r（P < 0.05）. Using RNRf/RNRr and CLas-4G/HLBr primer sets to perform duplex real-time PCR，it was found that RNRf/RNRr + CLas-4G/HLBr duplex real-time PCR was more sensitive than single primer real-time PCR. Therefore，RNRf/RNRr + CLas-4G/HLBr duplex real-time PCR could be used as an early diagnostic tool in low incidence areas of citrus Huanglongbing.

Key words: citrus Huanglongbing, real-time PCR, primer, combined diagnoses

中图分类号:

S666

李镇希, 潘睿翾, 许美容, 郑正, 邓晓玲. 柑橘黄龙病菌双重实时荧光PCR检测方法的建立[J]. 园艺学报, 2023, 50(1): 188-196.

LI Zhenxi, PAN Ruixuan, XU Meirong, ZHENG Zheng, DENG Xiaoling. Development of Duplex Real-time PCR Assay of‘Candidatus Liberibacter asiatics’[J]. Acta Horticulturae Sinica, 2023, 50(1): 188-196.

图/表 7

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引物名称 Primer	引物序列（5′-3′） Primer sequence	参考文献 Reference
HLBas/HLBr	TCGAGCGCGTATGCAATACG/GCGTTATCCCGTAGAAAAAGGTAG	Li et al.，2006
CQULA04F/CQULA04R	TGGAGGTGTAAAAGTTGCCAAA/CCAACGAAAAGATCAGATATTCCTCTA	Wang et al.，2006
f-rplLAs/r-rplLAs	CGCCCGTTTCCGTTGT/AGCCTCTTTAAGCCCTAAATCAG	Teixeira et al.，2008
LJ900f/LJ900r	GCCGTTTTAACACAAAAGATGAATATC/ATAAATCAATTTGTTCTAGTTTACGAC	Morgan et al.，2012
RNRf/RNRr	CATGCTCCATGAAGCTACCC/GGAGCATTTAACCCCACGAA	Zheng et al.，2016
CLas-4G/HLBr	AGTCGAGCGCGTATGCGAAT/GCGTTATCCCGTAGAAAAAGGTAG	Bao et al.，2020
4CPf/4CPr	GCCAAAAGTCTTGCACGTAA/GCGAAAACTTACCGCCTCTA	Bao et al.，2020

引物名称 Primer	引物序列（5′-3′） Primer sequence	参考文献 Reference
HLBas/HLBr	TCGAGCGCGTATGCAATACG/GCGTTATCCCGTAGAAAAAGGTAG	Li et al.，2006
CQULA04F/CQULA04R	TGGAGGTGTAAAAGTTGCCAAA/CCAACGAAAAGATCAGATATTCCTCTA	Wang et al.，2006
f-rplLAs/r-rplLAs	CGCCCGTTTCCGTTGT/AGCCTCTTTAAGCCCTAAATCAG	Teixeira et al.，2008
LJ900f/LJ900r	GCCGTTTTAACACAAAAGATGAATATC/ATAAATCAATTTGTTCTAGTTTACGAC	Morgan et al.，2012
RNRf/RNRr	CATGCTCCATGAAGCTACCC/GGAGCATTTAACCCCACGAA	Zheng et al.，2016
CLas-4G/HLBr	AGTCGAGCGCGTATGCGAAT/GCGTTATCCCGTAGAAAAAGGTAG	Bao et al.，2020
4CPf/4CPr	GCCAAAAGTCTTGCACGTAA/GCGAAAACTTACCGCCTCTA	Bao et al.，2020

引物名称 Primer name	曲线下面积 Area under curve	约登指数 Youden index	临界Ct值 Cut-off Ct value	敏感性/% Sensitivity	特异性/% Specificity	似然比 Likelihood ratio		预测值/% Predictive value		检出率/% Detection rate
引物名称 Primer name	曲线下面积 Area under curve	约登指数 Youden index	临界Ct值 Cut-off Ct value	敏感性/% Sensitivity	特异性/% Specificity	阳性 Positive	阴性 Negative	阳性 Positive	阴性 Negative	检出率/% Detection rate
HLBas/HLBr	0.957	0.8006	32.08	83.02	97.04	28.02	0.17	91.7	93.6	25.53
CQULA04F/ CQULA04R	0.963	0.8572	33.36	88.68	97.04	29.93	0.12	92.2	92.2	27.13
f-rplLAs/ r-rplLAs	0.933	0.8498	36.74	88.68	96.30	23.94	0.12	90.4	95.6	27.66
LJ900f/LJ900r	0.954	0.8194	33.00	84.91	97.04	28.66	0.16	91.8	94.2	26.06
RNRf/RNRr	0.971	0.8498	30.19	88.68	96.30	23.94	0.12	90.4	95.6	28.19
CLas-4G/HLBr	0.966	0.8349	32.44	88.68	94.81	17.10	0.12	87.0	95.5	28.72
4CPf/4CPr	0.964	0.8457	30.96	86.79	97.78	39.06	0.14	93.9	93.9	26.06

引物名称 Primer name	曲线下面积 Area under curve	约登指数 Youden index	临界Ct值 Cut-off Ct value	敏感性/% Sensitivity	特异性/% Specificity	似然比 Likelihood ratio		预测值/% Predictive value		检出率/% Detection rate
引物名称 Primer name	曲线下面积 Area under curve	约登指数 Youden index	临界Ct值 Cut-off Ct value	敏感性/% Sensitivity	特异性/% Specificity	阳性 Positive	阴性 Negative	阳性 Positive	阴性 Negative	检出率/% Detection rate
HLBas/HLBr	0.957	0.8006	32.08	83.02	97.04	28.02	0.17	91.7	93.6	25.53
CQULA04F/ CQULA04R	0.963	0.8572	33.36	88.68	97.04	29.93	0.12	92.2	92.2	27.13
f-rplLAs/ r-rplLAs	0.933	0.8498	36.74	88.68	96.30	23.94	0.12	90.4	95.6	27.66
LJ900f/LJ900r	0.954	0.8194	33.00	84.91	97.04	28.66	0.16	91.8	94.2	26.06
RNRf/RNRr	0.971	0.8498	30.19	88.68	96.30	23.94	0.12	90.4	95.6	28.19
CLas-4G/HLBr	0.966	0.8349	32.44	88.68	94.81	17.10	0.12	87.0	95.5	28.72
4CPf/4CPr	0.964	0.8457	30.96	86.79	97.78	39.06	0.14	93.9	93.9	26.06

串联检测引物 Tandem assay primer	曲线下面积 Area under curve	敏感性/% Sensitivity	特异性/% Specificity	似然比 Likelihood ratio		预测值/% Predictive value
串联检测引物 Tandem assay primer	曲线下面积 Area under curve	敏感性/% Sensitivity	特异性/% Specificity	阳性 Positive	阴性 Negative	阳性 Positive	阴性 Negative
CQULA04F/CQULA04R + RNRf/RNRr	0.957	81.48	99.25	109.19	0.19	97.8	93.0
CQULA04F/CQULA04 + CLas-4G/HLBr	0.958	81.48	98.51	54.59	0.19	95.7	93.0
CQULA04F/CQULA04R + 4CPf/4CPr	0.956	81.48	98.51	54.59	0.19	95.7	93.0
RNRf/RNRr + CLas-4G/HLBr	0.963	85.19	99.25	114.15	0.15	97.9	94.3
RNRf/RNRr + 4CPf/4CPr	0.961	83.33	99.25	111.67	0.17	97.8	93.7
CLas-4G/HLBr + 4CPf/4CPr	0.960	83.33	97.76	37.22	0.17	93.7	93.6