Development of Duplex Real-time PCR Assay of‘Candidatus Liberibacter asiatics’

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (1): 188-196.doi: 10.16420/j.issn.0513-353x.2021-0502

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Development of Duplex Real-time PCR Assay of‘Candidatus Liberibacter asiatics’

LI Zhenxi, PAN Ruixuan, XU Meirong, ZHENG Zheng, DENG Xiaoling^*()

Citrus Huanglongbing Reseach Laboratory，South China Agricultural University，Guangzhou 510642，China

Received:2022-04-25 Revised:2022-06-17 Online:2023-01-25 Published:2023-01-18
Contact: *（E-mail:xldeng@scau.edu.cn）

Abstract

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

CLC Number:

S666

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.

Figures/Tables 7

References 29

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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

Development of Duplex Real-time PCR Assay of‘Candidatus Liberibacter asiatics’

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并联检测引物 Parallel 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
并联检测引物 Parallel 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	0.966	92.59	94.03	15.51	0.079	86.2	96.9
CQULA04F/CQULA04 + CLas-4G/HLBr	0.971	92.59	93.28	13.79	0.079	86.2	96.9
CQULA04F/CQULA04R + 4CPf/4CPr	0.972	88.89	96.27	23.82	0.120	90.6	95.6
RNRf/RNRr + CLas-4G/HLBr	0.957	88.89	91.79	10.83	0.120	81.4	95.3
RNRf/RNRr + 4CPf/4CPr	0.959	87.04	94.78	16.66	0.140	87.0	94.8
CLas-4G/HLBr + 4CPf/4CPr	0.959	87.04	94.78	16.66	0.140	87.0	94.8

模板稀释倍数 Relative template concentration	RNRf/RNRr	CLas-4G/HLBr	RNRf/RNRr + CLas-4G/HLBr
10⁰	17.64	17.67	16.53
10¹	20.97	20.97	20.01
10²	24.34	24.46	23.38
10³	27.54	28.00	26.64
10⁴	31.40	31.41	30.28
10⁵	34.94	35.27	34.50
10⁶	N/A	N/A	37.21
ddH₂O	N/A	N/A	N/A

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[13]	BEN Hai-yan1，SHI Yan-xia2，XIE Xue-wen2，CHAI A-li2，and LI Bao-ju2,*. Studies of Soil Improvement Effect of Calcium Cyanamide and Its Control#br# Efficiency on Soil-borne Diseases of Vegetable Crops [J]. ACTA HORTICULTURAE SINICA, 2016, 43(11): 2173-2181.
[14]	FU Li-li，WU Ju-xun，and YI Hua-lin*. Expression Analysis of Citrate Metabolic Related Genes in Late-ripening Mutant of Navel Orange and Its Wild Type [J]. ACTA HORTICULTURAE SINICA, 2016, 43(1): 38-46.
[15]	LI Jia-wei，LIN Ming，HU Hong，WANG Xiao-xuan，GUO Yan-mei，HUANG Ze-jun，DU Yong-chen，and GAO Jian-chang**. The Combined Effects of Different TYLCV Resistant Genes and Temperatures on TYLCV Duplication [J]. ACTA HORTICULTURAE SINICA, 2016, 43(1): 71-79.