| 段玉,马志敏,许建建,宾羽,宋震,周常勇.柑橘黄龙病菌的重组酶聚合酶扩增(RPA)检测[J].中国南方果树,2021,50(5): |
| 柑橘黄龙病菌的重组酶聚合酶扩增(RPA)检测 |
| Establishment of RPA detection system for citrus Huanglongbing |
| 投稿时间：2021-01-13 修订日期：2021-03-03 |
| DOI：10.13938/j.issn.1007-1431.20210021 |
| 中文关键词: 柑橘黄龙病,重组酶聚合酶恒温扩增(RPA),快速检测 |
| 英文关键词:Citrus Huanglongbing Recombinase polymerase amplification (RPA) Rapid detection |
| 基金项目:基金项目： 国家重点研发计划（2017YFD0202002） |
|
| 摘要点击次数: 1800 |
| 全文下载次数: 323 |
| 中文摘要: |
| 【目的】本研究旨在建立黄龙病菌的重组酶聚合酶扩增(Recombinase Polymerase Amplification,RPA)快速检测体系,丰富柑橘黄龙病的高效简便检测方法。【方法】以黄龙病菌株psy62的保守基因序列设计并筛选特异性引物,经灵敏度比较和检测特异性验证,建立该病的RPA检测体系。经PCR、RPA和实时荧光PCR对87份柑橘样品的黄龙病菌检测,验证RPA检测体系的适用性。【结果】建立了黄龙病菌的RPA检测方法。(1)特异性：能特异性检测柑橘黄龙病。(2)灵敏度：RPA是PCR的100倍,与实时荧光PCR相当。(3)检测体系：只需20min,大幅提高检测效率。(4)适用性：RPA与实时荧光PCR检出率均为35.63%,比PCR的31.03%略高。【结论】建立了黄龙病菌的RPA检测方法,具有特异性强、灵敏度高、检测快速、无需特殊仪器设备和操作简便等特点,为柑橘黄龙病提供了高效简便的快速检测手段。 |
| 英文摘要: |
| Establishment of RPA detection system for citrus Huanglongbing
DUAN Yu, MA Zhimin, XU Jianjian, BIN Yu, SONG Zhen, ZHOU Changyong
(Citrus Research Institute of Southwest University/Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing 400712,China)
Abstract:【Objective】Citrus Huanglongbing caused by Candidatus Liberibacter spp.is a serious threat to citrus industry. Candidatus Liberibacter asiaticus (CLas) is the main pathogen in China. The rapid and accurate detection of CLas is the basis of field prevention and laboratory research. To establish Recombinase Polymerase Amplification (RPA) by electrophoresis detection system and overcome the shortcomings of fluorescence RPA detection method in fluorescence judgment. Fluorescence RPA detection method uses recombinant polymerase after the crude extraction of citrus nucleic acid to conduct constant temperature rapid amplification of the gene fragment, adds diluted fluorescence reagent to complete visual detection of CLas within 15min. However, this method is not effective in many experiments in our laboratory, which showed that the background fluorescence of healthy control and water control was strong. When the concentration of nucleic acid of CLas was low, the subjective deviation of detection personnel would appear in the determination of fluorescence. Electrophoretic RPA detection is the optimization of fluorescence RPA detection, to avoid the uncertainty of the judgment of fluorescence RPA detection, and can provides an efficient and convenient method for the rapid detection of CLas.【Methods】According to the single copy of tufB-secE-nusG-rplKAJL-rpoB gene and 5 copies of Ribonucleotide-diphosphate reductase subunit beta (nrdB) sequence in the whole genome of Citrus Huanglongbing strain psy62 complete genome (NC_012985.3), designed 5 pairs of primers for RPA to detect CLas. In the primer screening system, the primers needs to be accurately tested the positive control, without nonspecific reactions on healthy control and water control. Send the RPA reaction products to The Beijing Genomics Institute BGI for sequence identification and sequence alignment with 6 of Citrus Huanglongbing strains randomly selected in NCBI. After primer screening, the RPA electrophoresis detection system of CLas was established. Compared the sensitivity of RPA, PCR and Real-time PCR, three detection methods were used to detect CLas positive nucleic acids diluted in multiple of 10. Verify the detection specificity of RPA, other citrus pathogens, including Xanthomonas citri subsp(Xcc), Citrus exocortis viroid(CEVd), Citrus tatter leaf virus(CTLV), Citrus tatter leaf virus(CTLV), Citrus tristeza virus(CTV) and Satsuma dwarf virus(SDV) were used for RPA detection. Total of 87 citrus samples from Sichuan, Yunnan, Jiangxi and Guangxi regions were tested by PCR, RPA and Real-time PCR to verify the applicability of the RPA. 【Results】Obtained the primers RPAHF5/RPAHR5 from 5 pairs of primers tested that could specifically detection the CLas, the sequencing result was consistent with the sequences of 6 Citrus Huanglongbing strains randomly selected on NCBI. The RPA detection system of CLas was established on this basis, the detection results can be clearly presented on agarose gel electrophoresis. The RPA detection system: nucleic acid 2.5μL, each of RPAHF5/RPAHR5 2.4μL, primer-free rehydration buffer 29.5μL, 280 mM magnesium acetate(MgOAc)2.5μL, add water to make up 50μL and shake to mix, after centrifuge keep 20mins at 39℃, add 50μL trichloromethane and mix, centrifugation 2mins, absorb 10ul supernatant products electrophoresis with 2.5% agar gel electrophoresis. Compared the sensitivity of the three detection systems , the RPA has a high detection sensitivity ,the sensitivity of RPA was 100 times that of PCR, which was the same as that of Real-time PCR. Detection specificity verification showed the RPA had strong specificity for CLas detection, while other citrus pathogens, including Xcc, CEVd, CTLV, CTLV, CTV and SDV were tested negative. The detection results of 87 samples collected from 4 provinces showed that the positive rate (35.63%) detected by RPA was consistent with that by Real-time PCR, 4.60% higher than 31.03% by PCR. The difference between the three detection methods was reflected in the 18 hybrid citrus samples from Sichuan, both RPA and Real-time PCR tested positive, while PCR tested 17 positive and 1 negative; in 11 sweet orange samples and 10 Eulika lemon samples from Yunnan, both RPA and Real-time PCR tested positive for 7 and 3 respectively, PCR tested positive for 6 and 1 respectively. On other citrus sample detection, the results of the three detection methods were consistent. The results showed that the RPA detection methods was stable and reliable for CLas detection. The RPA detection only needed low constant temperature and short reaction time, 20mins at 39℃, and was simple to operate and did not need complex instruments and equipment.【Conclusion】Established and optimized the RPA detection system, which has advantages of short time consuming only needs 20mins at 39℃. Compared with PCR and Real-time PCR, RPA was 100 times that of PCR and was same as that of Real-time PCR. The RPA detection was strong specificity for CLas, other citrus pathogens tested negative. The RPA system for CLas detection has advantages of short time consuming and detection with strong specificity, high sensitivity and low operating difficulty. It provides an efficient and convenient method for the rapid detection of citrus Huanglongbing.
Keywords: Citrus Huanglongbing; Recombinase polymerase amplification (RPA); Rapid detection |
| 查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
