Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification

J.B.W.J. Cornelissen*, A. De Greeff, A.E. Heuvelink, M. Swarts, H.E. Smith, F.J. Van der Wal

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

13 Citations (Scopus)

Abstract

A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12 ng/assay) and ∼60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4 × 104 cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

Original languageEnglish
Pages (from-to)4270-4281
JournalJournal of Dairy Science
Volume99
Issue number6
DOIs
Publication statusPublished - 2016

Fingerprint

Streptococcus uberis
raw milk
Streptococcus
Milk
assays
detection limit
Mastitis
DNA
diagnostic specificity
loop-mediated isothermal amplification
diagnostic sensitivity
mastitis
pretreatment
methodology
Enzymes
cows
sampling
milk
Genes

Keywords

  • Loop-mediated isothermal amplification (LAMP)
  • Mastitis
  • Raw milk
  • Streptococcus uberis

Cite this

@article{44a57897e3f44d14855d3eb71015cf6d,
title = "Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification",
abstract = "A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12 ng/assay) and ∼60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4 × 104 cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0{\%} and a diagnostic specificity of 96.0{\%}. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.",
keywords = "Loop-mediated isothermal amplification (LAMP), Mastitis, Raw milk, Streptococcus uberis",
author = "J.B.W.J. Cornelissen and {De Greeff}, A. and A.E. Heuvelink and M. Swarts and H.E. Smith and {Van der Wal}, F.J.",
year = "2016",
doi = "10.3168/jds.2015-10683",
language = "English",
volume = "99",
pages = "4270--4281",
journal = "Journal of Dairy Science",
issn = "0022-0302",
publisher = "American Dairy Science Association",
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Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification. / Cornelissen, J.B.W.J.; De Greeff, A.; Heuvelink, A.E.; Swarts, M.; Smith, H.E.; Van der Wal, F.J.

In: Journal of Dairy Science, Vol. 99, No. 6, 2016, p. 4270-4281.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification

AU - Cornelissen, J.B.W.J.

AU - De Greeff, A.

AU - Heuvelink, A.E.

AU - Swarts, M.

AU - Smith, H.E.

AU - Van der Wal, F.J.

PY - 2016

Y1 - 2016

N2 - A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12 ng/assay) and ∼60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4 × 104 cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

AB - A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12 ng/assay) and ∼60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4 × 104 cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

KW - Loop-mediated isothermal amplification (LAMP)

KW - Mastitis

KW - Raw milk

KW - Streptococcus uberis

U2 - 10.3168/jds.2015-10683

DO - 10.3168/jds.2015-10683

M3 - Article

VL - 99

SP - 4270

EP - 4281

JO - Journal of Dairy Science

JF - Journal of Dairy Science

SN - 0022-0302

IS - 6

ER -