Rapid Detection of Viable <i>Penicillium expansum</i> in Apple by PMA-qPCR

YANG Jin; DING Yu; YU Lüjian; FAN Yingying; LIU Fengjuan; ZHAO Xueqi; JIAO Ziwei; WANG Cheng

doi:10.13386/j.issn1002-0306.2022100153

Volume 44 Issue 16

Aug. 2023

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Science and Technology of Food Industry > 2023 > 44(16): 331-338. > DOI: 10.13386/j.issn1002-0306.2022100153

YANG Jin, DING Yu, YU Lüjian, et al. Rapid Detection of Viable Penicillium expansum in Apple by PMA-qPCR[J]. Science and Technology of Food Industry, 2023, 44(16): 331−338. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022100153.

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Rapid Detection of Viable Penicillium expansum in Apple by PMA-qPCR

YANG Jin^{1, 2,},
DING Yu^{1, 2},
YU Lüjian^{1, 2},
FAN Yingying²,
LIU Fengjuan²,
ZHAO Xueqi³,
JIAO Ziwei^1, ,,
WANG Cheng^2, ,

1.
College of Bio-and Geo-Sciences, Yili Normal University, Ili 835000, China
2.
Institute of Quality Standards & Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Key Laboratory of Functional Nutrition and Health of Characteristic Agricultural Products in Desert Oasis Ecological Region (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Laboratory of Quality and Safety Risk Assessment for Agro-Products (Urumqi), Ministry of Agriculture and Rural Affairs, Key Laboratory of Agro-products Quality and Safety of Xinjiang, Urumqi 830091, China
3.
College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi 830052, China

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Received Date: October 17, 2022
Available Online: June 14, 2023

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Abstract

Abstract

Objective: To establish a rapid detection method for viable Penicillium expansum by propidium monoazide (PMA) combined with quantitative real-time polymerase chain reaction (qPCR). Methods: PMA-qPCR detection method for viable P. expansum was established, including the optimization of the treatment concentration, dark incubation and exposure time of PMA, the screening of specific primers of P. expansum, and the conduction of qPCR. In addition, the standard curve was constructed, which was applied to the detection of artificially contaminated apples samples. The reliability of this method was also evaluated by comparing with the plate counting method. Results: The optimal PMA treatment conditions were: 10 µg/mL for PMA concentration, 5 min for the dark incubation and 10 min for the exposure time. Among the 4 pairs of primers, Pexp-patF showed strong specificity for P. expansum, which could be used as a optimal primer for PMA-qPCR detection. The correlation coefficient of the established quantitative standard curve was 0.9948, and the detection limit of the method was 10^2.6 CFU/mL. There was no obvious difference between the detection result of this method and the plate counting method, and the viable P. expansum could be detected in the non-rotted part of apples. Conclusion: The established PMA-qPCR technique could be applied to the detection of P. expansum in apples, which would provide technical support for the prevention and control of P. expansum.
- Penicillium expansum,
- propidium monoazide,
- quantitative real-time polymerase chain reaction,
- apple,
- viable cell

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[1]	LI Y, ZHANG X, NIE J, et al. Occurrence and co-occurrence of mycotoxins in apple and apple products from China[J]. Food Control,2020,118:107354. doi: 10.1016/j.foodcont.2020.107354
[2]	李勇, 单硕, 吴丹舟, 等. 棒曲霉素的毒性及其毒性机制的研究进展[J/OL]. 食品科学: 1−12[2022-10-14]. http://kns.cnki.net/kcms/detail/11.2206.TS.20220728.1846.030.html 1−12 LI Y, SHAN S, WU D Z, et al. Research progress on toxicity and toxicity mechanism of patulin[J/OL]. Food Science: 1−12[2022-10-14]. http://kns.cnki.net/kcms/detail/11.2206.TS.20220728.1846.030.html 1−12.
[3]	IMAN S I. The characteristics, occurrence, and toxicological effects of patulin[J]. Food & Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association,2019,129:301−311.
[4]	COTON M, BREGIER T, POIRIER E, et al. Production and migration of patulin in Penicillium expansum molded apples during cold and ambient storage[J]. International Journal of Food Microbiology,2020,313:108177.
[5]	WEI D M, XU J, DONG F S, et al. Penicillium and patulin distribution in pears contaminated with Penicillium expansum. Determination of patulin in pears by UHPLCMS/MS[J]. Journal of Integrative Agriculture,2017,16(7):1645−1651. doi: 10.1016/S2095-3119(16)61543-5
[6]	ATOUI A, KHOURY A E, KALLASSY M, et al. Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize[J]. International Journal of Food Microbiology,2012,154(1-2):59−65. doi: 10.1016/j.ijfoodmicro.2011.12.022
[7]	TANNOUS J, ATOUI A, KHOURY A E, et al. Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples[J]. Food Microbiology,2015,50:28−37. doi: 10.1016/j.fm.2015.03.001
[8]	RODRIGUEZ A, LUQUE M I, ANDRADE M J, et al. Development of real-time PCR methods to quantify patulin-producing molds in food products[J]. Food Microbiology,2011,28(6):1190−1199. doi: 10.1016/j.fm.2011.04.004
[9]	袁英哲, 韩剑, 王岩, 等. 梨火疫病菌活菌快速定量检测方法的建立[J]. 果树学报,2020,37(9):1425−1433. [YUAN Y Z, HAN J, WANG Y, et al. Establishment of rapid quantitative detection of viable Erwinia amylovora[J]. Journal of Fruit Science,2020,37(9):1425−1433. YUAN Y Z, HAN J, WANG Y, et al. Establishment of rapid quantitative detection of viable Erwinia amylovora[J]. Journal of Fruit Science, 2020, 37(9): 1425-1433.
[10]	NOCKER A, CAMPER A K. Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques[J]. Fems Microbiology Letters,2009,291(2):137−142. doi: 10.1111/j.1574-6968.2008.01429.x
[11]	王帅, 徐进, 许景升, 等. PMA-qPCR定量检测青枯菌活菌方法的建立[J]. 植物保护,2018,44(6):122−128. [WANG S, XU J, XU J S, et al. Development of a PMA-qPCR method for quantitative detection of Ralstonia solanacearum[J]. Plant Protection,2018,44(6):122−128. WANG S, XU J, XU J S, et al. Development of a PMA-qPCR method for quantitative detection of Ralstonia solanacearum[J]. Plant Protection, 2018, 44(6): 122-128.
[12]	张静, 陈曦, 王彬, 等. 改良单叠氮丙啶-荧光定量PCR法的建立及其检测抗结核药物活性的价值[J]. 中国防痨杂志,2020,42(5):472−480. [ZHANG J, CHEN X, WANG B, et al. Establishment of modified propidium monoazide (PMAxx)-quantitative PCR assay and its application for identification of antituberculosis drug activity[J]. Chinese Journal of Antituberculosis,2020,42(5):472−480. ZHANG J, CHEN X, WANG B, et al. Establishment of modified propidium monoazide (PMAxx)-quantitative PCR assay and its application for identification of antituberculosis drug activity[J]. Chinese Journal of Antituberculosis, 2020, 42(5): 472-480.
[13]	HU L, FU Y D, ZHANG S, et al. An assay combining droplet digital PCR with propidium monoazide treatment for the accurate detection of live cells of Vibrio vulnificus in plasma samples[J]. Frontiers in Microbiology,2022,13:927285. doi: 10.3389/fmicb.2022.927285
[14]	柯振华. 餐饮食品中6病原菌叠氮丙啶-定量聚合酶链反应检测方法开发与检验数据分析[J]. 食品安全质量检测学报,2020,11(19):6855−6861. [KE Z H. Development of detection method and test data analysis of 6 pathogenic bacteria azidopropidine-quantitative polymerase chain reaction detection method in catering food[J]. Journal of Food Safety & Quality,2020,11(19):6855−6861. KE Z H. Development of detection method and test data analysis of 6 pathogenic bacteria azidopropidine-quantitative polymerase chain reaction detection method in catering food[J]. Journal of Food Safety & Quality, 2020, 11(19): 6855-6861.
[15]	TANG M L Y, LAU S C K. Strategy to evaluate changes in bacterial community profiles and bacterial pathogen load reduction after sewage disinfection[J]. Frontiers in Microbiology,2022,13:919207. doi: 10.3389/fmicb.2022.919207
[16]	曹学仁, 周益林. 基于PCR技术的植物病原菌分子定量检测技术研究进展[J]. 植物保护,2020,46(4):7−11. [CAO X R, ZHOU Y L. Research progress of quantitative detection of plant pathogens using PCR technique[J]. Plant Protection,2020,46(4):7−11. CAO X R, ZHOU Y L. Research progress of quantitative detection of plant pathogens using PCR technique[J]. Plant Protection, 2020, 46(4): 7-11.
[17]	于璇, 王卫芳, 李献锋, 等. PMA-qPCR检测十字花科黑斑病菌活菌方法的建立[J]. 植物检疫,2021,35(4):49−54. [YU X, WANG W F, LI X F, et al. PMA-qPCR assay for the detection of viable Pseudomonas syringae pv.maculicola[J]. Plant Quarantine,2021,35(4):49−54. YU X, WANG W F, LI X F, et al. PMA-qPCR assay for the detection of viable Pseudomonas syringae pv. maculicola[J]. Plant Quarantine, 2021, 35(4): 49-54.
[18]	张丹丹, 许晓丽, 李健强, 等. PMA-qPCR检测两种丁香假单胞菌活性研究[C]//成都: 中国植物病理学会2019年学术年会论文集, 2019: 379 ZHANG D D, XU X L, LI J Q, et al. PMA-qPCR detection of two species of Pseudomonas syringae activity[C]//Chengdu: Proceedings of the 2019 Annual Conference of the Chinese Society of Plant Pathology, 2019: 379.
[19]	CRESPO-SEMPERE A, ESTIARTE N, MARIN S, et al. Propidium monoazide combined with real-time quantitative PCR to quantify viable Alternaria spp. contamination in tomato products[J]. International Journal of Food Microbiology,2013,165(3):214−220. doi: 10.1016/j.ijfoodmicro.2013.05.017
[20]	樊明涛, 毕静莹, 刘邻渭, 等. 浓缩苹果汁中扩展青霉菌实时PCR快速检测条件的优化[J]. 西北农林科技大学学报(自然科学版),2007(11):84−89. [FAN M T, BI J Y, LIU L W, et al. Condition optimization of real-time PCR used in rapid detection of Penicillium expansum in apple juice concentrate[J]. Journal of Northwest A&F University (Natural Science Edition),2007(11):84−89. FAN M T, BI J Y, LIU L W, et al. Condition optimization of real-time PCR used in rapid detection of Penicillium expansum in apple juice concentrate[J] Journal of Northwest A & F University (Natural Science Edition), 2007(11): 84-89.
[21]	赵利娜, 李慧芳, 余江, 等. 扩展青霉侵染柑橘机制分析(英文)[J]. 食品科学,2019,40(16):97−106. [ZHAO L N, LI H F, YU J, et al. A mechanistic study of citrus infection by Penicillium expansum[J]. Food Science,2019,40(16):97−106. ZHAO L N, LI H F, YU J, et al. A mechanistic study of citrus infection by Penicillium expansum[J]. Food Science, 2019, 40(16): 97-106.
[22]	何鸿举, 焦凌霞, 樊明涛, 等. 应用PCR技术快速检测腐烂苹果中扩展青霉菌[J]. 食品科学,2011,32(12):183−187. [HE H J, JIAO L X, FAN M T, et al. Rapid detection of Penicillium expansum in rotten apples by polymerase chain reaction[J]. Food Science,2011,32(12):183−187. HE H J, JIAO L X, FAN M T, et al. Rapid detection of Penicillium expansum in rotten apples by polymerase chain reaction[J]. Food Science, 2011, 32(12): 183-187.
[23]	尉冬梅, 徐军, 董丰收, 等. 大久保桃扩展青霉病斑外延组织中青霉菌及展青霉素的扩散范围检测[J]. 农产品质量与安全,2016(4):35−39. [WEI D M, XU J, DONG F S, et al. Detection of the spread range of Penicillium and patulin in the epitaxy tissue of Okubo peach extended Penicillium[J]. Quality and Safety of Agro-Products,2016(4):35−39. WEI D M, XU J, DONG F S, et al. Detection of the spread range of Penicillium and Patulin in the epitaxy tissue of Okubo peach extended Penicillium[J]. Quality and Safety of Agro-Products, 2016, (4): 35-39.
[24]	王艳玲, 郭小洁, 张紊玮, 等. 棒曲霉素生物合成及分子调控研究进展[J]. 食品科学,2020,41(17):267−274. [WANG Y L, GUO X J, ZHANG W W, et al. Recent advances in patulin biosynthesis and its molecular regulation[J]. Food Science,2020,41(17):267−274. WANG Y L, GUO X J, ZHANG W W, et al. Recent advances in patulin biosynthesis and its molecular regulation[J]. Food Science, 2020, 41(17): 267-274.
[25]	LÜ X C, LI Y, QIU W W, et al. Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine[J]. Food Control,2016,66:69−78. doi: 10.1016/j.foodcont.2016.01.040
[26]	王银环, 郑小玲, 阮昊, 等. 叠氮溴化丙锭与实时荧光定量PCR结合检测地衣芽孢杆菌活菌制品中金黄色葡萄球菌[J]. 中国药学杂志,2022,57(3):231−236. [WANG Y H, ZHEN X L, RUAN H, et al. Detection ofStaphylococcus aureus inBacillus licheniformis live products by PMA-qPCR[J]. Chinese Pharmaceutical Journal,2022,57(3):231−236. WANG Y H, ZHEN X L, RUAN H, et al. Detection of Staphylococcus aureus in Bacillus licheniformis live products by PMA-qPCR[J]. Chinese Pharmaceutical Journal, 2022, 57(03): 231-236.
[27]	FRISCH L M, MANN M A, MAREK D N, et al. Development and optimization of a loop-mediated isothermal amplification (LAMP) assay for the species-specific detection of Penicillium expansum[J]. Food Microbiology,2021,95:103681. doi: 10.1016/j.fm.2020.103681
[28]	中华人民共和国卫生部. GB 4789.15-2010食品安全国家标准食品微生物学检验霉菌和酵母计数[S]. 北京: 中国标准出版社, 2012: 151−158. Ministry of Health of the Ministry of Health of the People's Republic of China. GB 4789.15-2010 National Food Safety Standard for Microbiological Examination of Food-Count of mold and yeast[S]. Beijing: China Standards Publishing House, 2012: 151−158.
[29]	DIAS C O, SCARIOT M C, AMBONI R, et al. Application of propidium monoazide coupled with quantitative PCR to evaluate cell viability of Bifidobacterium animalis subsp. lactis in a non-dairy probiotic beverage[J]. Annals of Microbiology,2020,70:22. doi: 10.1186/s13213-020-01566-9
[30]	BARRETTA C, VERRUCK S, MARAN B M, et al. Listeria monocytogenes survival in raw Atlantic salmon (Salmo salar) fillet under in vitro simulated gastrointestinal conditions by culture, qPCR and PMA-qPCR detection methods[J]. LWT-Food Science and Technology,2019,107:132−137. doi: 10.1016/j.lwt.2019.03.015

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Science and Technology of Food Industry

Rapid Detection of Viable Penicillium expansum in Apple by PMA-qPCR

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