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Journal of clinical microbiology2023; 61(3); e0137522; doi: 10.1128/jcm.01375-22

Development and Application of Real-Time PCR Assay for Detection of Salmonella Abortusequi.

Abstract: Salmonella enterica subsp. serovar Abortusequi is a major pathogen in horse and donkey herds, causing abortion in pregnant equids and resulting in enormous economic losses. A rapid and reliable method is urgently needed to detect . Abortusequi in herds where the disease is suspected. To achieve this goal, a TaqMan-based real-time PCR assay targeting the gene for the flagellin protein phase 2 antigen FljB was developed. This real-time PCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the assay was 30 copies/μL of standard plasmid and 10 CFU/μL of bacterial DNA. Furthermore, 540 clinical samples, including 162 tissue, 192 plasma, and 186 vaginal swab samples collected between 2018 and 2021 in China, were tested to assess the performance of the developed assay. Compared to the gold standard method of bacterial isolation, the real-time PCR assay exhibited 100% positive agreement for all tissue, plasma and vaginal swab tests. Additionally, this assay detected DNA from Abortusequi from 56.7% (34/60) culture-negative tissue and 22.9% (41/179) culture-negative vaginal swab samples from infected equids. Receiver operating characteristic analysis demonstrated that the results of the developed real-time PCR assays were in significant agreement with those of the culture method. The real-time PCR assay can be completed within 45 min of extraction of DNA from samples. Our results show that this assay could serve as a reliable tool for the rapid detection of Abortusequi in tissue, plasma, and vaginal swab clinical samples.
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Publication Date: 2023-03-01 PubMed ID: 36856425PubMed Central: PMC10035326DOI: 10.1128/jcm.01375-22Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research is about the development of a rapid and reliable method, known as a Taqman-based real-time PCR assay, for detecting Salmonella enterica, a major bacterial pathogen in horse and donkey herds, that frequently results in pregnancy loss and consequent economic losses.

Understanding the Problem

  • The study’s primary purpose was to address the issue of Salmonella enterica subsp. serovar Abortusequi, a serious bacterial infection in horse and donkey herds that often leads to abortion in pregnant animals, resulting in significant economic losses.
  • The detection of this bacteria has been challenging due to the lack of a rapid and reliable method. Hence, there was an urgent need to develop an efficient testing method that was both accurate and quick.

Method Developed

  • The authors developed a TaqMan-based real-time PCR (Polymerase Chain Reaction) assay for this purpose. This sophisticated technology targets the gene responsible for the second phase of the flagellin protein antigen, FljB, found in the bacteria.
  • The real-time PCR assay demonstrated high specificity, sensitivity, and reproducibility, meaning it was reliable in detecting the bacteria accurately.
  • The detection limit of the assay was notably low, capturing 30 copies/μL of standard plasmid and 10 CFU/μL of bacterial DNA. This high sensitivity is significant as it means the test can identify even small amounts of the bacteria.

Performance Assessment of the Assay

  • The researchers tested 540 clinical samples that included tissue, plasma, and vaginal swab samples, collected between 2018 and 2021 in China.
  • The results of the real-time PCR assay were compared to the gold standard method – bacterial isolation. In all tissue, plasma, and vaginal swab tests, the PCR assay displayed 100% positive agreement, establishing the reliability of the test.
  • The test could also detect bacterial DNA in 56.7% of culture-negative tissue and 22.9% of culture-negative vaginal swab samples, from infected equids.
  • The Receiver operating characteristic analysis, a statistical technique used to evaluate the performance of diagnostic tests, indicated that the results of the real-time PCR assays were significantly in agreement with those of the culture method.

Speed and Application

  • The real-time PCR assay can be completed within 45 minutes of extracting DNA from samples, demonstrating the test’s efficiency and speed.
  • The researchers concluded that this assay could serve as a reliable tool for rapidly detecting Salmonella enterica subsp. serovar Abortusequi in tissue, plasma, and vaginal swab clinical samples from horses and donkeys.

Cite This Article

APA
Wang J, Guo K, Li S, Liu D, Chu X, Wang Y, Guo W, Du C, Wang X, Hu Z. (2023). Development and Application of Real-Time PCR Assay for Detection of Salmonella Abortusequi. J Clin Microbiol, 61(3), e0137522. https://doi.org/10.1128/jcm.01375-22

Publication

Journal of clinical microbiology
ISSN: 1098-660X
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 61
Issue: 3
Pages: e0137522
PII: e01375-22

Researcher Affiliations

Wang, Jinhui
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Guo, Kui
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Li, Shuaijie
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Liu, Diqiu
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Chu, Xiaoyu
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Wang, Yaoxin
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Guo, Wei
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Du, Cheng
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Wang, Xiaojun
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Hu, Zhe
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

MeSH Terms

  • Pregnancy
  • Female
  • Animals
  • Horses / genetics
  • Salmonella enterica / genetics
  • Real-Time Polymerase Chain Reaction
  • Reproducibility of Results
  • Salmonella Infections, Animal / diagnosis
  • Salmonella Infections, Animal / microbiology
  • Salmonella / genetics
  • DNA, Bacterial / genetics
  • Sensitivity and Specificity

Conflict of Interest Statement

The authors declare no conflict of interest.

References

This article includes 30 references
  1. Gal-Mor O. Persistent Infection and Long-Term Carriage of Typhoidal and Nontyphoidal Salmonellae.. Clin Microbiol Rev 2019 Jan;32(1).
    doi: 10.1128/CMR.00088-18pmc: PMC6302356pubmed: 30487167google scholar: lookup
  2. Jazeela K, Chakraborty A, Karunasagar I, Deekshit VK. Nontyphoidal Salmonella: a potential anticancer agent.. J Appl Microbiol 2020 Jan;128(1):2-14.
    doi: 10.1111/jam.14297pubmed: 31038778google scholar: lookup
  3. Besser JM. Salmonella epidemiology: A whirlwind of change.. Food Microbiol 2018 May;71:55-59.
    doi: 10.1016/j.fm.2017.08.018pubmed: 29366469google scholar: lookup
  4. Sahu B, Singh SD, Behera BK, Panda SK, Das A, Parida PK. Rapid detection of Salmonella contamination in seafoods using multiplex PCR.. Braz J Microbiol 2019 Jul;50(3):807-816.
    doi: 10.1007/s42770-019-00072-8pmc: PMC6863201pubmed: 31006836google scholar: lookup
  5. Issenhuth-Jeanjean S, Roggentin P, Mikoleit M, Guibourdenche M, de Pinna E, Nair S, Fields PI, Weill FX. Supplement 2008-2010 (no. 48) to the White-Kauffmann-Le Minor scheme.. Res Microbiol 2014 Sep;165(7):526-30.
    doi: 10.1016/j.resmic.2014.07.004pubmed: 25049166google scholar: lookup
  6. Xiong D, Song L, Geng S, Tao J, An S, Pan Z, Jiao X. One-Step PCR Detection of Salmonella Pullorum/Gallinarum Using a Novel Target: The Flagellar Biosynthesis Gene flhB.. Front Microbiol 2016;7:1863.
    doi: 10.3389/fmicb.2016.01863pmc: PMC5118417pubmed: 27920764google scholar: lookup
  7. Grandolfo E, Parisi A, Ricci A, Lorusso E, de Siena R, Trotta A, Buonavoglia D, Martella V, Corrente M. High mortality in foals associated with Salmonella enterica subsp. enterica Abortusequi infection in Italy.. J Vet Diagn Invest 2018 May;30(3):483-485.
    doi: 10.1177/1040638717753965pmc: PMC6505811pubmed: 29322884google scholar: lookup
  8. Wang H, Liu KJ, Sun YH, Cui LY, Meng X, Jiang GM, Zhao FW, Li JJ. Abortion in donkeys associated with Salmonella abortus equi infection.. Equine Vet J 2019 Nov;51(6):756-759.
    doi: 10.1111/evj.13100pubmed: 30868638google scholar: lookup
  9. Uzzau S, Brown DJ, Wallis T, Rubino S, Leori G, Bernard S, Casadesús J, Platt DJ, Olsen JE. Host adapted serotypes of Salmonella enterica.. Epidemiol Infect 2000 Oct;125(2):229-55.
    doi: 10.1017/s0950268899004379pmc: PMC2869595pubmed: 11117946google scholar: lookup
  10. Barrandeguy ME, Carossino M. Infectious diseases in donkeys and mules: an overview and update. J Equine Vet Sci 65:98–105.
    doi: 10.1016/j.jevs.2018.02.026google scholar: lookup
  11. Niwa H, Hobo S, Kinoshita Y, Muranaka M, Ochi A, Ueno T, Oku K, Hariu K, Katayama Y. Aneurysm of the cranial mesenteric artery as a site of carriage of Salmonella enterica subsp. enterica serovar Abortusequi in the horse.. J Vet Diagn Invest 2016 Jul;28(4):440-4.
    doi: 10.1177/1040638716649640pubmed: 27271985google scholar: lookup
  12. McQuiston JR, Parrenas R, Ortiz-Rivera M, Gheesling L, Brenner F, Fields PI. Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella.. J Clin Microbiol 2004 May;42(5):1923-32.
    doi: 10.1128/JCM.42.5.1923-1932.2004pmc: PMC404605pubmed: 15131150google scholar: lookup
  13. Chiang YC, Wang HH, Ramireddy L, Chen HY, Shih CM, Lin CK, Tsen HY. Designing a biochip following multiplex polymerase chain reaction for the detection of Salmonella serovars Typhimurium, Enteritidis, Infantis, Hadar, and Virchow in poultry products.. J Food Drug Anal 2018 Jan;26(1):58-66.
    doi: 10.1016/j.jfda.2016.11.019pmc: PMC9332633pubmed: 29389589google scholar: lookup
  14. Liu R, Wang Z, Liu X, Chen A, Yang S. Rapid on-site detection of Salmonella pullorum based on lateral flow nucleic acid assay combined with recombinase polymerase amplification reaction.. Poult Sci 2020 Dec;99(12):7225-7232.
    doi: 10.1016/j.psj.2020.10.020pmc: PMC7705040pubmed: 33248640google scholar: lookup
  15. Ye Q, Shang Y, Chen M, Pang R, Li F, Xiang X, Wang C, Zhou B, Zhang S, Zhang J, Yang X, Xue L, Ding Y, Wu Q. Identification of Novel Sensitive and Reliable Serovar-Specific Targets for PCR Detection of Salmonella Serovars Hadar and Albany by Pan-Genome Analysis.. Front Microbiol 2021;12:605984.
    doi: 10.3389/fmicb.2021.605984pmc: PMC8011537pubmed: 33815306google scholar: lookup
  16. Zhou YY, Kang XL, Meng C, Xiong D, Xu Y, Geng SZ, Pan ZM, Jiao XA. Multiple PCR assay based on the cigR gene for detection of Salmonella spp. and Salmonella Pullorum/Gallinarum identification.. Poult Sci 2020 Nov;99(11):5991-5998.
    doi: 10.1016/j.psj.2020.07.026pmc: PMC7647733pubmed: 33142517google scholar: lookup
  17. Xu Y, Hu Y, Guo Y, Zhou Z, Xiong D, Meng C, Li Q, Geng S, Pan Z, Jiao X. A new PCR assay based on the new gene-SPUL_2693 for rapid detection of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum.. Poult Sci 2018 Nov 1;97(11):4000-4007.
    doi: 10.3382/ps/pey254pubmed: 30101343google scholar: lookup
  18. Xiong D, Song L, Tao J, Zheng H, Zhou Z, Geng S, Pan Z, Jiao X. An Efficient Multiplex PCR-Based Assay as a Novel Tool for Accurate Inter-Serovar Discrimination of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin.. Front Microbiol 2017;8:420.
    doi: 10.3389/fmicb.2017.00420pmc: PMC5352712pubmed: 28360901google scholar: lookup
  19. El Houmami N, Durand GA, Bzdrenga J, Darmon A, Minodier P, Seligmann H, Raoult D, Fournier PE. A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene of Kingella kingae and Application to 201 Pediatric Clinical Specimens.. J Clin Microbiol 2018 Aug;56(8).
    doi: 10.1128/JCM.00505-18pmc: PMC6062779pubmed: 29875189google scholar: lookup
  20. Kasturi KN, Drgon T. Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples.. Appl Environ Microbiol 2017 Jul 15;83(14).
    doi: 10.1128/AEM.00644-17pmc: PMC5494621pubmed: 28500041google scholar: lookup
  21. Heymans R, Vila A, van Heerwaarden CAM, Jansen CCC, Castelijn GAA, van der Voort M, Biesta-Peters EG. Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR.. PLoS One 2018;13(10):e0206316.
    doi: 10.1371/journal.pone.0206316pmc: PMC6201931pubmed: 30359449google scholar: lookup
  22. Traub-Dargatz JL, Garber LP, Fedorka-Cray PJ, Ladely S, Ferris KE. Fecal shedding of Salmonella spp by horses in the United States during 1998 and 1999 and detection of Salmonella spp in grain and concentrate sources on equine operations.. J Am Vet Med Assoc 2000 Jul 15;217(2):226-30.
    doi: 10.2460/javma.2000.217.226pubmed: 10909464google scholar: lookup
  23. Yang Y, Zhang J, Zhu C, Meng X, Sun S, Zhu G. A promising detection candidate for flagellated Salmonella spp.. AMB Express 2019 Aug 14;9(1):128.
    doi: 10.1186/s13568-019-0851-0pmc: PMC6694378pubmed: 31414324google scholar: lookup
  24. El-Sayed Ahmed K A, El-Shishtawy M, El-Taweel F, El-Mansoury H. Multiplex PCR for Diagnosis of Salmonella enterica Serovar Typhi.. Clin Lab 2015;61(10):1537-43.
    doi: 10.7754/clin.lab.2015.150115pubmed: 26642717google scholar: lookup
  25. Sonne-Hansen J, Jenabian SM. Molecular serotyping of Salmonella: identification of the phase 1 H antigen based on partial sequencing of the fliC gene.. APMIS 2005 May;113(5):340-8.
    doi: 10.1111/j.1600-0463.2005.apm_113505.xpubmed: 16011660google scholar: lookup
  26. Kavlick MF. Development of a universal internal positive control.. Biotechniques 2018 Nov;65(5):275-280.
    doi: 10.2144/btn-2018-0034pubmed: 30394127google scholar: lookup
  27. Xiong D, Song L, Pan Z, Jiao X. Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay.. Front Microbiol 2018;9:1718.
    doi: 10.3389/fmicb.2018.01718pmc: PMC6079294pubmed: 30108571google scholar: lookup
  28. Ben Hassena A, Barkallah M, Fendri I, Grosset N, Ben Neila I, Gautier M, Gdoura R. Real time PCR gene profiling and detection of Salmonella using a novel target: The siiA gene.. J Microbiol Methods 2015 Feb;109:9-15.
    doi: 10.1016/j.mimet.2014.11.018pubmed: 25486551google scholar: lookup
  29. Wang M, Yang J, Gai Z, Huo S, Zhu J, Li J, Wang R, Xing S, Shi G, Shi F, Zhang L. Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk.. Int J Food Microbiol 2018 Feb 2;266:251-256.
    doi: 10.1016/j.ijfoodmicro.2017.12.011pubmed: 29272725google scholar: lookup
  30. Teh CSJ, Lau MY, Chong CW, Ngoi ST, Chua KH, Lee WS, Thong KL. One-step differential detection of Salmonella enterica serovar Typhi, serovar Paratyphi A and other Salmonella spp. by using a quadruplex real-time PCR assay.. J Microbiol Methods 2021 Apr;183:106184.
    doi: 10.1016/j.mimet.2021.106184pubmed: 33662480google scholar: lookup

Citations

This article has been cited 10 times.
  1. Gao W, Liu M, Nurdaly K, Caidan D, Sun Y, Duan J, Zhao J, Gong X, Zhou J, Zhang Y, Chen Q. Unveiling Equine Abortion Pathogens: A One Health Perspective on Prevalence and Resistance in Northwest China. Pathogens 2025 Dec 11;14(12).
    doi: 10.3390/pathogens14121275pubmed: 41471229google scholar: lookup
  2. Thapa S, Chen FC. Quantitative Detection of Salmonella Typhimurium in Ground Chicken Using a Surface Plasmon Resonance (SPR) Biosensor. Biosensors (Basel) 2025 Dec 15;15(12).
    doi: 10.3390/bios15120814pubmed: 41440295google scholar: lookup
  3. Bakishev T, Amirgazin A, Kuibagarov M, Shevtsov A, Bakisheva Z, Yessembekova G, Kairzhanova A, Kadyrov A, Guo K, Wang X, Abdrakhmanov S, Borovikov S. Genome-wide characterization and comparative phylogenomics of three Salmonella Abortusequi strains isolated from equine abortions in Kazakhstan. Vet World 2025 Jun;18(6):1571-1580.
    doi: 10.14202/vetworld.2025.1571-1580pubmed: 40689182google scholar: lookup
  4. Guo K, Zhang Z, Yang Y, Zhang W, Wang J, Li S, Chu X, Guo W, Liu D, Wang Y, Hu Z, Wang X. Development and Application of an iELISA for the Detection of Antibody against Salmonella Abortusequi. Transbound Emerg Dis 2023;2023:1403180.
    doi: 10.1155/2023/1403180pubmed: 40303659google scholar: lookup
  5. Cao S, Kong X, Liu Y, Wang Z, Zhang Z, Lei X, Li P, Wang L, Yang F, Liu S, Li R, Li Y, Feng X, Wang T. Characterization and genomic analysis of Salmonella Abortusequi phage, vB_SalP_LDDK01, and its biocontrol application in donkey meat. Front Cell Infect Microbiol 2024;14:1527201.
    doi: 10.3389/fcimb.2024.1527201pubmed: 39764155google scholar: lookup
  6. Zhang Y, Guo X, Yu M, Sun L, Qu Y, Guo K, Hu Z, Liu D, Zhang H, Wang X. Equine ANP32 proteins support influenza A virus RNA polymerase activity. Virol Sin 2023 Oct 27;38(6):951-60.
    doi: 10.1016/j.virs.2023.10.009pubmed: 39491182google scholar: lookup
  7. Manikandan R, Rajagunalan S, Malmarugan S, Gupta C. First report on whole genome sequencing and comparative genomics of Salmonella enterica serovar Abortusequi isolated from Donkey in India. Sci Rep 2024 Oct 8;14(1):23455.
    doi: 10.1038/s41598-024-73904-9pubmed: 39379477google scholar: lookup
  8. Braun SD, Müller E, Frankenfeld K, Gary D, Monecke S, Ehricht R. A Proof-of-Concept Protein Microarray-Based Approach for Serotyping of Salmonella enterica Strains. Pathogens 2024 Apr 25;13(5).
    doi: 10.3390/pathogens13050355pubmed: 38787207google scholar: lookup
  9. Guo K, Guo W, Liu D, Zhang W, Yang Y, Zhang Z, Li S, Wang J, Chu X, Wang Y, Hu Z, Wang X. Development and application of a competitive ELISA for the detection of antibodies against Salmonella Abortusequi in equids. J Clin Microbiol 2023 Nov 21;61(11):e0027323.
    doi: 10.1128/jcm.00273-23pubmed: 37874302google scholar: lookup
  10. Borovikov S, Ryskeldina A, Tursunov K, Syzdykova A, Akibekov O. Recombinant Salmonella enterica OmpX protein expression and its potential for serologically diagnosing Salmonella abortion in mares. Vet World 2023 Sep;16(9):1790-1795.
    doi: 10.14202/vetworld.2023.1790-1795pubmed: 37859952google scholar: lookup
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