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Article(id=1276601095468217136, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276600957765021779, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2024.06.004, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1684425600000, receivedDateStr=2023-05-19, revisedDate=1687622400000, revisedDateStr=2023-06-25, acceptedDate=null, acceptedDateStr=null, onlineDate=1782295019260, onlineDateStr=2026-06-24, pubDate=1719244800000, pubDateStr=2024-06-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782295019260, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782295019260, creator=13701087609, updateTime=1782295019260, updator=13701087609, issue=Issue{id=1276600957765021779, tenantId=1146029695717560320, journalId=1235980609244409860, year='2024', volume='45', issue='6', pageStart='1095', pageEnd='1302', issueExtLink='null', onlineDate='null', pubDate='1719244800000', pubDateStr='2024-06-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782294986430, creator='13701087609', updateTime=1782348406834, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276825019267285043, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276600957765021779, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276825019271479348, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276600957765021779, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1120, endPage=1126, ext={EN=ArticleExt(id=1276601095845704498, articleId=1276601095468217136, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Establishment of TaqMan Probe Real-time Fluorescent Quantitative PCR Detection Method for Areca Palm Yellow Leaf Phytoplasma, columnId=1236256430337085821, journalTitle=Chinese Journal of Tropical Crops, columnName=Omics & Biotechnology, runingTitle=null, highlight=null, articleAbstract=

Areca palm is an important tropical economic crop in Hainan Province. Areca palm yellow leaf disease (YLD) caused by phytoplasma infection is a devastating disease in the production of areca in China. In order to establish an accurate and efficient detection method for areca palm yellow leaf phytoplasma, this study designed and synthesized specific primers AMf/AMr and AM-Prode based on the 16S rDNA gene of areca palm yellow leaf phytoplasma. The method was used to test the accuracy, sensitivity, specificity and repeatability, and to detect phytoplasma diseases in other plants. The method could accurately detect the positive samples, and the healthy samples had no amplification curve. In the sensitivity test, the method could detect the sample concentration level of 1.16×101 copies/μL, and the standard curve equation was y=–3.4185x+43.624, the amplification efficiency was 96.12%, and the correlation coefficient R2=0.9833. In the specificity test, the method had good specificity for the detection of YLD, and the genomes of areca, other disease pathogens and the endophytes did not interfere with the method. In the repeatability test, the method had good repeatability for the detection of YLD. The detection method could be used to detect 8 phytoplasma diseases such as chinaberry yellow leaf disease, ‘Pericampylus glaucus’ witches' broom disease and pepper yellow leaf disease and the detection of phytoplasma has certain universality. The establishment of detection method is conducive to providing reliable technical means for the accurate diagnosis of YLD, pathogen monitoring and vector insect detection.

, authors=null, authorsList=Zhaowei LIN, Xiuli MENG, Qinghua TANG, Xiaoqing NIU, Weiwei SONG, authorCompany=null, correspAuthors=Weiwei SONG, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1276601099578635070, articleId=1276601095468217136, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=槟榔黄化植原体TaqMan探针实时荧光定量PCR检测方法的建立, columnId=1236256430517440904, journalTitle=热带作物学报, columnName=组学与生物技术, runingTitle=null, highlight=null, articleAbstract=

槟榔是海南省重要的热带经济作物，由植原体侵染引起的槟榔黄化病（areca palm yellow leaf disease，YLD）是当前我国槟榔生产上的一种毁灭性病害。为了建立精准高效的槟榔黄化植原体检测方法，本研究基于槟榔黄化植原体16S rDNA基因，设计并合成特异性引物AMf/AMr和探针AM-Prode，使用该方法进行准确性、敏感性、特异性及重复性测试，并在其他植物的植原体病害进行检测。结果显示：本检测方法能够准确的检测出阳性样品，健康样品无扩增曲线；在敏感性测试中，该检测方法能检测到1.16×101 copies/μL样本浓度水平，其标准曲线方程为y=–3.4185x + 43.624，扩增效率为96.12%，相关系数R2=0.9833；在特异性测试中，该检测方法对YLD的检测具有较好的特异性，槟榔、槟榔其他病害病原及其内生菌的基因组对本方法未造成干扰；在重复性测试中，该检测方法对槟榔黄化病的检测具有较好的重复性；并且该检测方法可对苦楝黄化病、细圆藤丛枝病及辣椒黄化病等8种植原体病害进行检测，对植原体的检测具有一定的通用性。本检测方法的建立，有利于为槟榔黄化病的精准诊断、病原监测及媒介昆虫的检测等研究提供可靠的技术手段。

, authors=

林兆威（1993—），男，硕士，研究实习员，研究方向：热带经济作物主要病害综合防治。

, authorsList=林兆威, 孟秀利, 唐庆华, 牛晓庆, 宋薇薇, authorCompany=null, correspAuthors=宋薇薇, authorNote=null, correspAuthorsNote=
* 宋薇薇（SONG Weiwei），E-mail：。
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林兆威（1993—），男，硕士，研究实习员，研究方向：热带经济作物主要病害综合防治。

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林兆威（1993—），男，硕士，研究实习员，研究方向：热带经济作物主要病害综合防治。

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Rapid and efficient detection of 16SrI group areca palm yellow leaf phytoplasma in China by loop-mediated isothermal amplification[J]. The Plant Pathology Journal, 2020, 36(5): 459-467., articleTitle=Rapid and efficient detection of 16SrI group areca palm yellow leaf phytoplasma in China by loop-mediated isothermal amplification, refAbstract=null), Reference(id=1276824450070876901, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, doi=null, pmid=null, pmcid=null, year=2022, volume=181, issue=2, pageStart=152, pageEnd=159, url=null, language=null, rfNumber=[18], rfOrder=30, authorNames=YU S S, ZHANG X C, SONG W W, QIN W Q, journalName=Annals of Applied Biology, refType=null, unstructuredReference=YU S S, ZHANG X C, SONG W W, QIN W Q. Accurate and sensitive detection of areca palm yellow leaf phytoplasma in China by droplet digital PCR targeting tuf gene sequence[J]. Annals of Applied Biology, 2022, 181(2): 152-159., articleTitle=Accurate and sensitive detection of areca palm yellow leaf phytoplasma in China by droplet digital PCR targeting tuf gene sequence, refAbstract=null), Reference(id=1276824450146374374, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, doi=null, pmid=null, pmcid=null, year=2022, volume=42, issue=11, pageStart=68, pageEnd=72, url=null, language=null, rfNumber=[19], rfOrder=31, authorNames=林兆威, 宋薇薇, 唐庆华, 牛晓庆, 王宇航, 孟秀利, journalName=热带农业科学, refType=null, unstructuredReference=林兆威, 宋薇薇, 唐庆华, 牛晓庆, 王宇航, 孟秀利. 槟榔种苗和采摘工具的植原体检测[J]. 热带农业科学, 2022, 42(11): 68-72., articleTitle=槟榔种苗和采摘工具的植原体检测, refAbstract=null), Reference(id=1276824450209288935, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, doi=null, pmid=null, pmcid=null, year=2022, volume=42, issue=11, pageStart=68, pageEnd=72, url=null, language=null, rfNumber=[19], rfOrder=32, authorNames=LIN Z W, SONG W W, TANG Q H, NIU X Q, WANG Y H, MENG X L, journalName=Chinese Journal of Tropical Agriculture, refType=null, unstructuredReference=LIN Z W, SONG W W, TANG Q H, NIU X Q, WANG Y H, MENG X L. Phytoplasma detection of areca palm seedling and picking tool[J]. Chinese Journal of Tropical Agriculture, 2022, 42(11): 68-72. 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1–5: YLD samples; 6: Negative samples; 7: Blank controls.

, figureFileSmall=yJBM0j49CG+qv2FDbgTw2w==, figureFileBig=j82LDVyuf3vPGpRLfHN9rg==, tableContent=null), ArticleFig(id=1276824442135253688, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=CN, label=图1, caption=TaqMan qPCR引物和探针的准确性验证

1~5：槟榔黄化病样品；6：阴性对照；7：空白对照。

, figureFileSmall=yJBM0j49CG+qv2FDbgTw2w==, figureFileBig=j82LDVyuf3vPGpRLfHN9rg==, tableContent=null), ArticleFig(id=1276824442537906873, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=EN, label=Fig. 2, caption=TaqMan qPCR sensitivity tests, figureFileSmall=WgZ2sPxoRkNtY81UGt1RYw==, figureFileBig=FTPHJRr78Ecyu+mm4JMn9A==, tableContent=null), ArticleFig(id=1276824442605015738, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=CN, label=图2, caption=TaqMan qPCR敏感性检测

1–8: 1.16×107, 1.16×106, 1.16×105, 1.16×104, 1.16×103, 1.16×102, 1.16×101, 1.16×100 copies/μL; 9: Negative control.

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1: CK+; 2-10: APV1, ANRSV, C. gloeosporioides, B. andropogonis, P. ananatis, S. yantingensis, C. albidum, B. cereus, F. australicus; 11: CK–.

, figureFileSmall=9eNRJ5Jv08pvlGcuxxarvg==, figureFileBig=SPdjnjL00lOWpA43oLlPHQ==, tableContent=null), ArticleFig(id=1276824444685390527, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=EN, label=Fig. 5, caption=TaqMan qPCR detection of phytoplasmas on different plants

1–8: chinaberry yellow leaf phytoplasma, Catharanthus roseus little leaf phytoplasma, Trema orientalis witches broom phytoplasma, pepper yellow leaf phytoplasma, Parthenium hysterophorus witches broom phytoplasma, Pericampylus glaucus witches broom phytoplasma, peanut witches broom phytoplasma and bamboo witches broom phytoplasma; 9-16: healthy samples corresponding to 8 phytoplasmas; 17: Blank control.

, figureFileSmall=gZepa2wiGBnfq2npLwsU+g==, figureFileBig=s9tmmtWSn9hY7sw0GYMrdw==, tableContent=null), ArticleFig(id=1276824444744110784, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=CN, label=图5, caption=TaqMan qPCR检测不同植物上的植原体

1~8：苦楝黄化植原体、长春花小叶植原体、山黄麻丛枝植原体、辣椒黄化植原体、银胶菊丛枝植原体、细圆藤丛枝植原体、花生丛枝植原体、竹子丛枝植原体；9~16为8种植原体对应的健康样品；17：空白对照。

, figureFileSmall=gZepa2wiGBnfq2npLwsU+g==, figureFileBig=s9tmmtWSn9hY7sw0GYMrdw==, tableContent=null), ArticleFig(id=1276824444827996865, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=EN, label=Tab. 1, caption=

Information of primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name序列（5'–3'）Sequence (5'–3')片段长度Fragment size/bp用途Purpose
P1AAGAGTTTGATCCTGGCTCAGGATT约1800巢氏PCR产物制作阳性质粒标准品
P7CGTCCTTCATCGGCTCTT
R16F2nGAAACGACTGCTAAGACT
R16R2TGACGGGCGGTGTGTACAAACCCCG约1200
AMfCCCAATGTGGCCGTTCAAC
AMrGAAACGACTGCTAAGACTGGATAGGA173TaqMan qPCR
AM-ProdeCCTACCAAGACTATGATGTG
), ArticleFig(id=1276824444890911426, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=CN, label=表1, caption=

本研究中所用引物信息

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name序列（5'–3'）Sequence (5'–3')片段长度Fragment size/bp用途Purpose
P1AAGAGTTTGATCCTGGCTCAGGATT约1800巢氏PCR产物制作阳性质粒标准品
P7CGTCCTTCATCGGCTCTT
R16F2nGAAACGACTGCTAAGACT
R16R2TGACGGGCGGTGTGTACAAACCCCG约1200
AMfCCCAATGTGGCCGTTCAAC
AMrGAAACGACTGCTAAGACTGGATAGGA173TaqMan qPCR
AM-ProdeCCTACCAAGACTATGATGTG
), ArticleFig(id=1276824444945437379, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=EN, label=Tab. 2, caption=

TaqMan fluorescence quantitative PCR repeated test

, figureFileSmall=null, figureFileBig=null, tableContent=
标准品浓度Plasmid standard concentration/(copies·μL–1)组内重复性试验Intra batch repeat test组间重复性试验Inter batch repeat test
平均值±标准差Mean±SD变异系数Coefficient of variation/%平均值±标准差Mean±SD变异系数Coefficient of variation/%
1.16×10527.53±0.160.5827.89±0.451.60
1.16×10430.62±0.280.9330.96±0.461.49
1.16×10333.69±0.260.7933.98±0.641.90
), ArticleFig(id=1276824445037712068, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276601095468217136, language=CN, label=表2, caption=

TaqMan荧光定量PCR重复测试

, figureFileSmall=null, figureFileBig=null, tableContent=
标准品浓度Plasmid standard concentration/(copies·μL–1)组内重复性试验Intra batch repeat test组间重复性试验Inter batch repeat test
平均值±标准差Mean±SD变异系数Coefficient of variation/%平均值±标准差Mean±SD变异系数Coefficient of variation/%
1.16×10527.53±0.160.5827.89±0.451.60
1.16×10430.62±0.280.9330.96±0.461.49
1.16×10333.69±0.260.7933.98±0.641.90
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槟榔黄化植原体TaqMan探针实时荧光定量PCR检测方法的建立
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林兆威 , 孟秀利 , 唐庆华 , 牛晓庆 , 宋薇薇 *
热带作物学报 | 组学与生物技术 2024,45(6): 1120-1126
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热带作物学报 |组学与生物技术 2024 , 45 (6) : 1120 -1126
槟榔黄化植原体TaqMan探针实时荧光定量PCR检测方法的建立
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林兆威, 孟秀利, 唐庆华, 牛晓庆, 宋薇薇*
作者信息
  • 中国热带农业科学院椰子研究所/海南省院士团队创新中心，海南文昌　571339
通讯作者:
* 宋薇薇（SONG Weiwei），E-mail：。
Establishment of TaqMan Probe Real-time Fluorescent Quantitative PCR Detection Method for Areca Palm Yellow Leaf Phytoplasma
Zhaowei LIN, Xiuli MENG, Qinghua TANG, Xiaoqing NIU, Weiwei SONG*
Affiliations
  • Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences / Hainan Innovation Center of Academician Team, Wenchang, Hainan 571339, China
出版时间: 2024-06-25 doi: 10.3969/j.issn.1000-2561.2024.06.004
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摘要
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槟榔是海南省重要的热带经济作物，由植原体侵染引起的槟榔黄化病（areca palm yellow leaf disease，YLD）是当前我国槟榔生产上的一种毁灭性病害。为了建立精准高效的槟榔黄化植原体检测方法，本研究基于槟榔黄化植原体16S rDNA基因，设计并合成特异性引物AMf/AMr和探针AM-Prode，使用该方法进行准确性、敏感性、特异性及重复性测试，并在其他植物的植原体病害进行检测。结果显示：本检测方法能够准确的检测出阳性样品，健康样品无扩增曲线；在敏感性测试中，该检测方法能检测到1.16×101 copies/μL样本浓度水平，其标准曲线方程为y=–3.4185x + 43.624，扩增效率为96.12%，相关系数R2=0.9833；在特异性测试中，该检测方法对YLD的检测具有较好的特异性，槟榔、槟榔其他病害病原及其内生菌的基因组对本方法未造成干扰；在重复性测试中，该检测方法对槟榔黄化病的检测具有较好的重复性；并且该检测方法可对苦楝黄化病、细圆藤丛枝病及辣椒黄化病等8种植原体病害进行检测，对植原体的检测具有一定的通用性。本检测方法的建立，有利于为槟榔黄化病的精准诊断、病原监测及媒介昆虫的检测等研究提供可靠的技术手段。

槟榔  /  槟榔黄化病  /  植原体  /  TaqMan实时荧光定量PCR  /  病害检测

Areca palm is an important tropical economic crop in Hainan Province. Areca palm yellow leaf disease (YLD) caused by phytoplasma infection is a devastating disease in the production of areca in China. In order to establish an accurate and efficient detection method for areca palm yellow leaf phytoplasma, this study designed and synthesized specific primers AMf/AMr and AM-Prode based on the 16S rDNA gene of areca palm yellow leaf phytoplasma. The method was used to test the accuracy, sensitivity, specificity and repeatability, and to detect phytoplasma diseases in other plants. The method could accurately detect the positive samples, and the healthy samples had no amplification curve. In the sensitivity test, the method could detect the sample concentration level of 1.16×101 copies/μL, and the standard curve equation was y=–3.4185x+43.624, the amplification efficiency was 96.12%, and the correlation coefficient R2=0.9833. In the specificity test, the method had good specificity for the detection of YLD, and the genomes of areca, other disease pathogens and the endophytes did not interfere with the method. In the repeatability test, the method had good repeatability for the detection of YLD. The detection method could be used to detect 8 phytoplasma diseases such as chinaberry yellow leaf disease, ‘Pericampylus glaucus’ witches' broom disease and pepper yellow leaf disease and the detection of phytoplasma has certain universality. The establishment of detection method is conducive to providing reliable technical means for the accurate diagnosis of YLD, pathogen monitoring and vector insect detection.

areca  /  areca palm yellow leaf disease  /  phytoplasma  /  TaqMan qPCR  /  disease detection
林兆威, 孟秀利, 唐庆华, 牛晓庆, 宋薇薇. 槟榔黄化植原体TaqMan探针实时荧光定量PCR检测方法的建立. 热带作物学报, 2024 , 45 (6) : 1120 -1126 . DOI: 10.3969/j.issn.1000-2561.2024.06.004
Zhaowei LIN, Xiuli MENG, Qinghua TANG, Xiaoqing NIU, Weiwei SONG. Establishment of TaqMan Probe Real-time Fluorescent Quantitative PCR Detection Method for Areca Palm Yellow Leaf Phytoplasma[J]. Chinese Journal of Tropical Crops, 2024 , 45 (6) : 1120 -1126 . DOI: 10.3969/j.issn.1000-2561.2024.06.004
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槟榔（Areca catechu L.）是棕榈科槟榔属多年生热带经济作物，中国的“四大南药”之首。在我国，槟榔栽培于海南、台湾及云南等地，其中海南省槟榔种植面积占全国总面积的95%以上，产量位居世界第二位[1]。目前，槟榔作为海南省重要的热带经济作物，是海南省政府重点发展的“六棵树”之一，也是海南省中东部地区230万农民收入的主要经济来源[2]。
由植原体侵染引起的槟榔黄化病（areca palm yellow leaf disease，YLD）是当前我国槟榔生产上的一种毁灭性病害。该病于1981年在我国的海南省屯昌县发生为害[3]，目前已蔓延至海南省各市县的槟榔种植区[4]。槟榔黄化病在槟榔生产上的为害逐年加重，轻者减产10%~20%，重者减产50%~60%，局部地区甚至毁种绝收。据不完全统计，每年因黄化病造成的经济损失高达20亿元以上，严重影响海南省农民脱贫致富和农村经济增长[5]。
精准的病原检测有利于对病害的诊断。TaqMan荧光定量PCR技术目前广泛应用于临床疾病诊断和动植物检验检疫上。为了建立精准高效的槟榔黄化植原体检测方法，本研究基于槟榔黄化植原体的16S rDNA基因序列设计特异性引物和探针，并对该方法的准确性、敏感性、特异性及重复性进行了验证，以期为YLD精准诊断、病原监测及媒介昆虫检测的研究提供可靠的技术手段。
1　材料与方法
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1.1　材料
1.1.1　供试样品
槟榔黄化植原体DNA通过通用引物P1/P7[6]与R16F2n/R16R2[7]巢氏PCR检测为阳性，槟榔长线形病毒1（Areca palm velarivirus 1，APV1）cDNA、槟榔坏死环斑病毒（Areca palm necrotic ringspot virus，ANRSV）cDNA分别通过RT-qPCR、RT-PCR检测为阳性[8-9]，上述样品由中国热带农业科学院椰子研究所提供；槟榔炭疽菌（c. gloeosporioides）、槟榔叶片分离的内生或致病细菌：Burkholderia andropogonis、Pantoea ananatis、Sphingomonas yantingensis、Curtobacterium albidum、Bacillus cereus、Frondihabitans australicus基因组DNA由中国热带农业科学院椰子研究所提供；健康槟榔叶片样品采自中国热带农业科学院椰子研究所槟榔苗圃；银胶菊丛枝植原体（16SrⅡ-A亚组）、长春花小叶植原体（16SrⅠ-B亚组）DNA由中国热带农业科学院环境与植物保护研究所馈赠；竹丛枝植原体（16SrI-B亚组）、苦楝黄化植原体（16SrI-B亚组）、细圆藤丛枝植原体（16SrⅠ-B亚组）、辣椒黄化植原体（16SrⅠ-B亚组）、山黄麻丛枝植原体（16SrXXXⅡ-D亚组）、花生丛枝植原体（16SrⅡ-A亚组）DNA及上述对应的健康样品DNA由中国热带农业科学院椰子研究所提供。
1.1.2　主要试剂与仪器
主要试剂：植物总DNA提取试剂盒、2×Taq PCR mix、琼脂糖凝胶回收试剂盒、质粒小提试剂盒及实时荧光定量PCR（探针法）试剂盒购自天根生物科技（北京）有限公司；pMDTM18-T Vector购自TaKaRa公司；DH5α感受态细胞购自北京全式金生物技术有限公司；其他试剂为国产分析纯。
主要仪器：5810R型台式冷冻离心机，Bio-Rad T100 PCR仪器，QuantStudio 3 Real-time定量PCR仪，超微量分光光度计Thermo ND2000。
1.2　方法
1.2.1　引物与探针设计及合成
根据槟榔黄化植原体16S rDNA基因序列（GenBank登录号：KF728948.1），利用Primer Express 3.0软件在该基因保守的区域设计荧光定量PCR特异性引物AMf/AMr和AM-Prode探针（表1）。合成植原体16S rDNA基因巢氏PCR通用引物P1/P7[6]与R16F2n/R16R2[7]用于构建阳性质粒标准品。引物与探针由生工生物工程（上海）股份有限公司合成，引物采用PAGE纯化，探针采用HPLC纯化。
1.2.2　DNA提取
取样品叶片0.1 g，参照植物总DNA提取试剂盒说明书进行DNA提取，并于–20 ℃保存。
1.2.3　实时荧光定量PCR验证引物和探针的准确性
使用引物AMf/AMr和探针AM-Prode对5份经巢氏PCR扩增测序的阳性样品进行实时荧光定量PCR，健康槟榔样品作为阴性对照，无菌无酶ddH2O作为空白对照。反应体系：2×SuperReal PreMix（Probe）10 μL，正、反向引物（10 μmol/L）各0.6 μL，荧光探针（10 μmol/L）0.4 μL，DNA模板2 μL，50×ROX Reference Dye*3 0.2 μL，加ddH2O至20 μL。反应程序：95 ℃预变性15 min；95 ℃变性3 s，60 ℃退火/延伸30 s，45个循环。
1.2.4　阳性质粒标准品的制备
使用第一步引物P1/P7与第二步引物R16F2n/R16R2，以阳性槟榔黄化病样品的DNA为模板，健康槟榔样品DNA阴性对照，ddH2O为空白对照进行巢式PCR，PCR产物进行琼脂糖凝胶电泳检测，切下目的条带，利用琼脂糖凝胶回收试剂盒进行目的条带纯化，纯化后产物连接到pMDTM18-T Vector，转化DH5α感受态细胞中，筛选并提取阳性克隆子的质粒，送生工生物工程（上海）股份有限公司进行测序。将测序正确的质粒使用超微量分光光度计测定浓度，置于–20 ℃保存备用。运用SHIRIMA等[10]的方法计算重组质粒中的拷贝数，重组质粒拷贝数（copies/μL）=（NA×C）/（109×重组质粒碱基对数×660 dalton/bp）。其中NA为阿伏伽德罗常数，6.02×1023；C为重组质粒的浓度（ng/μL）。最终计算得到重组质粒的拷贝数为1.16×1010 copies/μL。荧光定量PCR反应体系及程序参照1.2.3，巢式PCR反应体系及程序参照林兆威等[11]的方法。
1.2.5　敏感性测试与标准曲线的建立
以阳性质粒标准品为初始模板，梯度稀释出浓度依次为1.16×107、1.16×106、1.16×105、1.16×104、1.16×103、1.16×102、1.16×101、1.16×100 copies/μL。以此8个梯度浓度阳性质粒标准品为模板，健康槟榔样品为阴性对照，进行实时荧光定量PCR，反应体系及程序参照1.2.3。得到动力学扩增曲线和PCR扩增循环阈值（Ct）。运用Excel 2020软件，以Ct值为纵坐标，10为底质粒拷贝数的对数值为横坐标绘制标准曲线，获得回归直线方程，并计算该组体系的扩增效率：扩增效率（%）=10（–1/斜率）–1。
1.2.6　特异性测试
以槟榔黄化植原体的DNA为阳性对照（CK+），健康槟榔DNA为阴性对照（CK–），ANRSV、APV1的cDNA，槟榔炭疽菌、Burkholderia andropogonis、Pantoea ananatis、Sphingomonas yantingensis、Curtobacterium albidum、Bacillus cereus、Frondihabitans australicus的DNA为模板进行实时荧光定量PCR，检测其特异性，反应体系和程序参照1.2.3。
1.2.7　重复性测试
选取1.16×105、1.16×104、1.16×103 copies/μL三个浓度的阳性质粒标准品为模板，进行实时荧光定量PCR检测，每个浓度重复3次，共检测3次，进行组内重复性试验，计算Ct值的变异系数。同时，将上述浓度的阳性质粒标准品于–20 ℃保存3、6、9 d，以相同反应条件重复进行组间试验，计算Ct值的变异系数，验证该方法的重复性。反应体系和程序参照1.2.3。
1.2.8　不同植物植原体上的检测
对银胶菊丛枝植原体、竹丛枝植原体、长春花小叶植原体、苦楝黄化植原体、细圆藤丛枝植原体、辣椒黄化植原体、山黄麻丛枝植原体及花生丛枝植原体等8种不同植物的植原体DNA进行实时荧光定量PCR检测，对应的8种植物健康样品DNA为阴性对照，无酶无菌ddH2O为空白对照，反应体系和程序参照1.2.3。
2　结果与分析
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2.1　实时荧光定量PCR验证引物和探针的准确性
使用引物AMf/AMr和探针AM-Prode对阳性样品、健康槟榔样品进行TaqMan qPCR。如图1所示，阳性样品均出现“S”形扩增曲线，且曲线平滑，而阴性样品和空白对照未出现扩增曲线，说明该荧光定量PCR的引物/探针组合能检测到槟榔黄化植原体，可进一步验证该引物/探针组合其他特性。
2.2　敏感性测试与标准曲线的建立
将构建好的阳性质粒标准品浓度按10的倍数逐级稀释，以8个梯度浓度阳性质粒为模板，健康槟榔样品为阴性对照，进行TaqMan qPCR，并利用Ct值与质粒拷贝数的对数构建标准曲线。结果表明，该引物/探针组合能检测到1.16×101 copies/μL样本浓度水平（图2），标准曲线显示，Ct值与拷贝数的对数呈线性关系，其标准曲线方程为y=-3.4185x+43.624，扩增效率为96.12%，相关系数R2=0.9833（图3）。
2.3　特异性测试
使用槟榔黄化植原体和槟榔中主要的病害病原及内生菌进行TaqMan qPCR特异性验证，结果如图4所示，仅YLD出现扩增曲线，健康槟榔、APV1、ANRSV、槟榔炭疽菌、B. andropogonis、P. ananatis、S. yantingensis、C. albidum、B. cereus、F. australicus均未出现扩增曲线，说明该检测方法对YLD的检测具有较好的特异性，槟榔、槟榔其他病害病原及其内生菌的基因组对本方法未造成干扰。
2.4　重复性测试
选取浓度为1.16×105、1.16×104、1.16×103 copies/μL的阳性标准品为模板，进行TaqMan qPCR检测，分别验证该方法的组内和组间的重复性，结果如表2所示，组内变异系数小于1%，组间变异系数小于2%，该方法对YLD的检测具有较好的重复性。
2.5　不同植物植原体上的检测
如图5所示，供试的银胶菊丛枝植原体、竹丛枝植原体、长春花小叶植原体、苦楝黄化植原体、细圆藤丛枝植原体、辣椒黄化植原体、山黄麻丛枝植原体及花生丛枝植原体通过该TaqMan qPCR方法检测均为植原体阳性，而对应的健康样品无扩增曲线，说明本方法对植原体的检测具有一定的通用性，可用于检测上述植原体病害。
3　讨论
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在大多数植原体病害的检测中，常用巢氏PCR通用引物P1/P7[6]与R16F2n/R16R2[7]，并将R16F2n/R16R2扩增的全长序列进行虚拟限制性片段长度多态性（restriction fragment length polymorphism，RFLP）分析作为植原体鉴定的重要依据之一。由于上述巢氏PCR通用引物的敏感性较低，不利于低浓度病原的检测，因此，在植原体检测中常建立适用于该作物、该病害的检测技术。鹿鹏鹏等[12]基于16S rDNA基因建立了剑麻紫色卷叶病相关植原体单管巢式PCR检测方法，该检测技术最低检测限浓度为≥1 fg/μL；韩剑等[13]基于16S rDNA基因建立了枣疯病植原体TaqMan探针实时荧光定量PCR检测方法，该方法敏感性达60 copies/μL；张荣跃等[14]基于Sec A基因建立了甘蔗白叶病植原体PCR检测方法，该方法敏感性达50 fg/μL。不同的病原检测方法均具有优缺点，考虑方法的敏感性、特异性及重复性等问题的同时，也应考虑检测成本。如巢氏PCR检测成本低，但敏感性相对较低，荧光定量PCR检测所需设备昂贵，但敏感性较高。因此，针对不同作物、不同种类病原及其病害发病特点采取适宜的检测技术。如病原浓度高的植原体病害，可采取低成本的巢氏PCR检测技术即可完成绝大多数的检测需求。槟榔黄化病植原体具有分布不均匀[15]、浓度含量低的特点，因此所需敏感性较高的检测方法。目前，国内槟榔黄化植原体检测主要有巢氏PCR[16]、环介导等温扩增（loop mediated isothermal amplification，LAMP）[17]、实时荧光定量PCR[15]及微滴式数字PCR（droplet digital PCR，ddPCR）[18]等方法。巢氏PCR检测方法受限于敏感性的原因，因此未能在YLD检测中大量使用。国内槟榔黄化病LAMP检测技术的敏感性为200 ag/μL[17]，实时荧光定量PCR检测方法的敏感性为1.16×104 copies/μL[15]，而本研究建立的TaqMan qPCR敏感性达1.16×101 copies/μL（换算为50 ag/μL），敏感性高于上述2个检测方法。微滴式数字PCR敏感性达0.07 copies/μL[18]，且样本含量可绝对定量，但成本高、仪器设备昂贵，由于条件受限，目前尚未大量使用。
本检测方法所需的引物和探针对槟榔黄化植原体具有高度特异性，才能避免非特异性扩增，而槟榔黄化植原体16S rDNA基因与部分槟榔叶绿体基因组序列同源性较高。因此，本试验在槟榔黄化植原体16S rDNA基因保守区域，且与槟榔叶绿体基因组特异的位置设计引物和探针，经大量筛选，获得特异性引物AMf/AMr和探针AM-Prode。经测试，该检测方法具有较好的敏感性、特异性及重复性，满足于日常的槟榔黄化植原体检测。并且将该方法应用于苦楝黄化病、细圆藤丛枝病及辣椒黄化病等其他8种植原体病害中，能够区别健康和感病材料，说明本方法适用于其他植物植原体的检测，具有一定的通用性，可用于对其他植物植原体病害或疑似槟榔黄化病中间寄主进行初步诊断。
在应用方面，该方法实现了海南省17个市县的926分样品的黄化病检测，研究结果明确了植原体为海南省槟榔病理性黄化的主要病原之一[4]；并对槟榔种苗和槟榔采摘工具进行了检测，实现了一直以来由于种苗植原体含量较低造成检测难的问题[19]；在其他植物植原体病害的应用方面，对叶下珠黄化和丛枝症状样本进行初步的诊断，并发现叶下珠为植原体侵染的新寄主[11]。目前，该技术在本课题组已应用于植原体含量较低的疑似槟榔黄化病媒介昆虫的检测。
基金
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  • 海南省自然科学基金项目(321QN345)
  • 海南省院士创新平台科研专项(YSPTZX202138; YSPTZX202151)
文献
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2024年第45卷第6期
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doi: 10.3969/j.issn.1000-2561.2024.06.004
  • 接收时间：2023-05-19
  • 首发时间：2026-06-24
  • 出版时间：2024-06-25
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  • 收稿日期：2023-05-19
  • 修回日期：2023-06-25
基金
海南省自然科学基金项目(321QN345)
海南省院士创新平台科研专项(YSPTZX202138; YSPTZX202151)
作者信息
    中国热带农业科学院椰子研究所/海南省院士团队创新中心，海南文昌　571339

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* 宋薇薇（SONG Weiwei），E-mail：。
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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