An Improved Method for Quantification of Viable Cells in Infected Soil Products by Propidium Monoazide Coupled with Real-Time PCR

Overview

Journal Microorganisms

Specialty Microbiology

Date 2022 May 28

PMID 35630479

Authors

Lida Chen

Lei Li

Xuewen Xie

Ali Chai

Yanxia Shi

Tengfei Fan

Jianming Xie

Baoju Li

Affiliations

Soon will be listed here.

Abstract

is a soil-borne pathogen that causes root rot disease in cucumber. To date, quantitative real-time PCR (qPCR) is a common tool to detect the content of in soil. However, qPCR cannot distinguish between viable and nonviable cells. The aim of this study was to develop a detection technique to pretreat tissue fluid with propidium monoazide (PMA) followed by extract DNA, and then to quantify viable cells in contaminated soil. In this work, the specific primer pair F8-1/F8-2 was designed based on the translation elongation factor (EF) gene and a PMA-qPCR assay was established to amplify and quantify soils of viable cells. The PMA pretreatment test was optimized, which indicated that the optimal PMA concentration and light exposure time were 50 mmol L and 15 min, respectively. The lowest limit of viable cells in suspension detected and soil by PMA-qPCR were 82 spore mL and 91.24 spore g, respectively. For naturally contaminated soil, viable cells were detected in eight of the 18 samples, and the amount ranged from 10 to 10 spore g. In conclusion, the PMA-qPCR method has the characteristics of high sensitivity, efficiency, and time saving, which could support nursery plants to avoid infection and agro-industry losses.

Citing Articles

Advances in the detection technology of vegetable soil borne fungi and bacteria.

Chen L, Lu G, Yang S, Gong B, Lu Y, Wu X Front Microbiol. 2024; 15:1460729.

PMID: 39703705 PMC: 11656321. DOI: 10.3389/fmicb.2024.1460729.

Practical application of PMA-qPCR assay for determination of viable cells of inter-species biofilm of .

Kendra S, Czucz Varga J, Gaalova-Radochova B, Bujdakova H Biol Methods Protoc. 2024; 9(1):bpae081.

PMID: 39659667 PMC: 11631528. DOI: 10.1093/biomethods/bpae081.

Zearalenone contamination in maize, its associated producing fungi, control strategies, and legislation in Sub-Saharan Africa.

Hudu A, Addy F, Mahunu G, Abubakari A, Opoku N Food Sci Nutr. 2024; 12(7):4489-4512.

PMID: 39055180 PMC: 11266927. DOI: 10.1002/fsn3.4125.

MicroMPN: methods and software for high-throughput screening of microbe suppression in mixed populations.

Franco Melendez K, Schuster L, Donahey M, Kairalla E, Jansen M, Reisch C Microbiol Spectr. 2024; 12(3):e0357823.

PMID: 38353567 PMC: 10923211. DOI: 10.1128/spectrum.03578-23.

Rapid Monitoring of Viable Genetically Modified Using a Cell-Direct Quantitative PCR Method Combined with Propidium Monoazide Treatment.

Qin Y, Qu B, Lee B Microorganisms. 2023; 11(5).

PMID: 37317102 PMC: 10220751. DOI: 10.3390/microorganisms11051128.

References

Pastrana A, Basallote-Ureba M, Aguado A, Capote N . Potential Inoculum Sources and Incidence of Strawberry Soilborne Pathogens in Spain. Plant Dis. 2019; 101(5):751-760. DOI: 10.1094/PDIS-08-16-1177-RE. View

Li B, Liu Y, Shi Y, Xie X, Guo Y . First Report of Crown Rot of Grafted Cucumber Caused by Fusarium solani in China. Plant Dis. 2019; 94(11):1377. DOI: 10.1094/PDIS-03-10-0217. View

Meng X, Qi X, Han Z, Guo Y, Wang Y, Hu T . Latent Infection of Valsa mali in the Seeds, Seedlings and Twigs of Crabapple and Apple Trees is a Potential Inoculum Source of Valsa Canker. Sci Rep. 2019; 9(1):7738. PMC: 6533284. DOI: 10.1038/s41598-019-44228-w. View

Casasnovas F, Fantini E, Palazzini J, Giaj-Merlera G, Chulze S, Reynoso M . Development of amplified fragment length polymorphism (AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot. J Appl Microbiol. 2013; 114(6):1782-92. DOI: 10.1111/jam.12183. View

Elizaquivel P, Aznar R, Sanchez G . Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field. J Appl Microbiol. 2013; 116(1):1-13. DOI: 10.1111/jam.12365. View

Nocker A, Mazza A, Masson L, Camper A, Brousseau R . Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology. J Microbiol Methods. 2008; 76(3):253-61. DOI: 10.1016/j.mimet.2008.11.004. View

ODonnell K, Sutton D, Rinaldi M, Sarver B, Arunmozhi Balajee S, Schroers H . Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. J Clin Microbiol. 2010; 48(10):3708-18. PMC: 2953079. DOI: 10.1128/JCM.00989-10. View

Tian Q, Feng J, Hu J, Zhao W . Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide. Sci Rep. 2016; 6:35457. PMC: 5064318. DOI: 10.1038/srep35457. View

Chai A, Ben H, Guo W, Shi Y, Xie X, Li L . Quantification of Viable Cells of pv. in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay. Plant Dis. 2020; 104(8):2225-2232. DOI: 10.1094/PDIS-11-19-2397-RE. View

10.

Nocker A, Camper A . Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol. 2006; 72(3):1997-2004. PMC: 1393219. DOI: 10.1128/AEM.72.3.1997-2004.2006. View

11.

Pan Y, Breidt Jr F . Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol. 2007; 73(24):8028-31. PMC: 2168130. DOI: 10.1128/AEM.01198-07. View

12.

Nocker A, Cheung C, Camper A . Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods. 2006; 67(2):310-20. DOI: 10.1016/j.mimet.2006.04.015. View

13.

Golpayegani A, Douraghi M, Rezaei F, Alimohammadi M, Nabizadeh Nodehi R . Propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay for rapid detection of viable and viable but non-culturable (VBNC) in swimming pools. J Environ Health Sci Eng. 2019; 17(1):407-416. PMC: 6582174. DOI: 10.1007/s40201-019-00359-w. View

14.

Laidlaw A, Ganzle M, Yang X . Comparative assessment of qPCR enumeration methods that discriminate between live and dead Escherichia coli O157:H7 on beef. Food Microbiol. 2019; 79:41-47. DOI: 10.1016/j.fm.2018.11.002. View

15.

Vesper S, McKinstry C, Hartmann C, Neace M, Yoder S, Vesper A . Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA). J Microbiol Methods. 2007; 72(2):180-4. DOI: 10.1016/j.mimet.2007.11.017. View

16.

Josefsen M, Lofstrom C, Hansen T, Christensen L, Olsen J, Hoorfar J . Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Appl Environ Microbiol. 2010; 76(15):5097-104. PMC: 2916463. DOI: 10.1128/AEM.00411-10. View

17.

Elizaquivel P, Sanchez G, Selma M, Aznar R . Application of propidium monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water. Food Microbiol. 2012; 30(1):316-20. DOI: 10.1016/j.fm.2011.10.008. View

18.

Muraosa Y, Schreiber A, Trabasso P, Matsuzawa T, Taguchi H, Moretti M . Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC). Int J Med Microbiol. 2014; 304(3-4):505-11. DOI: 10.1016/j.ijmm.2014.03.001. View

19.

Zhu R, Li T, Jia Y, Song L . Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR. J Microbiol Methods. 2012; 90(3):262-6. DOI: 10.1016/j.mimet.2012.05.019. View

20.

Cawthorn D, Witthuhn R . Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide. J Appl Microbiol. 2008; 105(4):1178-85. DOI: 10.1111/j.1365-2672.2008.03851.x. View