Potential of orf12 region in TTS1 gene cluster of B. pseudomallei as specific marker for a sensitive Loop-Mediated Isothermal Amplification (LAMP) assay for melioidosis detection

K. Dzulaikha, Suhanawati A. Nur, Rahmah Mohd Amin

Research output: Contribution to journalArticle

Abstract

Introduction: Burkholderia pseudomallei is a gram-negative motile bacterium responsible for melioidosis, an endemic human disease across much of Southeast Asia and northern Australia. Due to clinical manifestations that frequently 'mimic' other disease, a rapid diagnosis for septicemic and acute melioidosis patients is much needed. Serological tests such as IHA and ELISA have poor sensitivity due to high background in seropositivity in endemic area while PCR and RT-PCR require sophisticated equipments make it almost impossible for rapid detection in rural district. As a rapid, simple and cost effective detection method, LAMP assay give a better anticipation for early detection of melioidosis. Objective: To define a highly specific region in B. pseudomallei for assessment of a highly sensitive LAMP assay for rapid diagnosis of melioidosis. Methods: Specific marker for B. pseudomallei was determined based on analysis and alignment of conserved region of orf12 in TTS1 gene cluster amongst all B. pseudomallei strains and its distinction from other Burkholderiae. Primers were designed using Primer Explorer software and the primers specificity were validated using Burkholderiae and other Bacteriacea. The sensitivity of primers to amplify target region was determined by defining the limit of detection (LOD) of optimized LAMP assay on 10-fold serial dilution of B. pseudomallei genomic DNA and B. pseudomallei suspension. Results & Discussion: The orf12 region of TTS1 gene cluster of B. pseudomallei LAMP assay showed high specificity towards B. pseudomallei strain. The orf12 LAMP assay of serially diluted B. pseudomallei genomic DNA gave a limit of detection (LOD) of 1.0 × 10-4 ng/μL while the LAMP assay of serially diluted B. pseudomallei suspension showed an LOD of 1.5 × 10-4 CFU/mL. Conclusion: High specificity and sensitivity of orf12 LAMP assay in this study proves that the orf12 region of TTS1 cluster gene in B. pseudomallei has the potential of being one of the DNA markers for rapid detection of melioidosis.

Original languageEnglish
Pages (from-to)241
Number of pages1
JournalAsian Pacific Journal of Tropical Disease
Volume4
Issue number3
DOIs
Publication statusPublished - Jun 2014

Fingerprint

Melioidosis
Burkholderia pseudomallei
Multigene Family
Burkholderia
Limit of Detection
Suspensions
Endemic Diseases
Polymerase Chain Reaction
Southeastern Asia
DNA
Serologic Tests
Gram-Negative Bacteria
Genetic Markers
Software

ASJC Scopus subject areas

  • Infectious Diseases
  • Microbiology (medical)

Cite this

@article{98de1642b48a48c886eb21bf44f992b6,
title = "Potential of orf12 region in TTS1 gene cluster of B. pseudomallei as specific marker for a sensitive Loop-Mediated Isothermal Amplification (LAMP) assay for melioidosis detection",
abstract = "Introduction: Burkholderia pseudomallei is a gram-negative motile bacterium responsible for melioidosis, an endemic human disease across much of Southeast Asia and northern Australia. Due to clinical manifestations that frequently 'mimic' other disease, a rapid diagnosis for septicemic and acute melioidosis patients is much needed. Serological tests such as IHA and ELISA have poor sensitivity due to high background in seropositivity in endemic area while PCR and RT-PCR require sophisticated equipments make it almost impossible for rapid detection in rural district. As a rapid, simple and cost effective detection method, LAMP assay give a better anticipation for early detection of melioidosis. Objective: To define a highly specific region in B. pseudomallei for assessment of a highly sensitive LAMP assay for rapid diagnosis of melioidosis. Methods: Specific marker for B. pseudomallei was determined based on analysis and alignment of conserved region of orf12 in TTS1 gene cluster amongst all B. pseudomallei strains and its distinction from other Burkholderiae. Primers were designed using Primer Explorer software and the primers specificity were validated using Burkholderiae and other Bacteriacea. The sensitivity of primers to amplify target region was determined by defining the limit of detection (LOD) of optimized LAMP assay on 10-fold serial dilution of B. pseudomallei genomic DNA and B. pseudomallei suspension. Results & Discussion: The orf12 region of TTS1 gene cluster of B. pseudomallei LAMP assay showed high specificity towards B. pseudomallei strain. The orf12 LAMP assay of serially diluted B. pseudomallei genomic DNA gave a limit of detection (LOD) of 1.0 × 10-4 ng/μL while the LAMP assay of serially diluted B. pseudomallei suspension showed an LOD of 1.5 × 10-4 CFU/mL. Conclusion: High specificity and sensitivity of orf12 LAMP assay in this study proves that the orf12 region of TTS1 cluster gene in B. pseudomallei has the potential of being one of the DNA markers for rapid detection of melioidosis.",
author = "K. Dzulaikha and Nur, {Suhanawati A.} and {Mohd Amin}, Rahmah",
year = "2014",
month = "6",
doi = "10.1016/S2222-1808(14)60547-8",
language = "English",
volume = "4",
pages = "241",
journal = "Asian Pacific Journal of Tropical Disease",
issn = "2222-1808",
publisher = "Elsevier BV",
number = "3",

}

TY - JOUR

T1 - Potential of orf12 region in TTS1 gene cluster of B. pseudomallei as specific marker for a sensitive Loop-Mediated Isothermal Amplification (LAMP) assay for melioidosis detection

AU - Dzulaikha, K.

AU - Nur, Suhanawati A.

AU - Mohd Amin, Rahmah

PY - 2014/6

Y1 - 2014/6

N2 - Introduction: Burkholderia pseudomallei is a gram-negative motile bacterium responsible for melioidosis, an endemic human disease across much of Southeast Asia and northern Australia. Due to clinical manifestations that frequently 'mimic' other disease, a rapid diagnosis for septicemic and acute melioidosis patients is much needed. Serological tests such as IHA and ELISA have poor sensitivity due to high background in seropositivity in endemic area while PCR and RT-PCR require sophisticated equipments make it almost impossible for rapid detection in rural district. As a rapid, simple and cost effective detection method, LAMP assay give a better anticipation for early detection of melioidosis. Objective: To define a highly specific region in B. pseudomallei for assessment of a highly sensitive LAMP assay for rapid diagnosis of melioidosis. Methods: Specific marker for B. pseudomallei was determined based on analysis and alignment of conserved region of orf12 in TTS1 gene cluster amongst all B. pseudomallei strains and its distinction from other Burkholderiae. Primers were designed using Primer Explorer software and the primers specificity were validated using Burkholderiae and other Bacteriacea. The sensitivity of primers to amplify target region was determined by defining the limit of detection (LOD) of optimized LAMP assay on 10-fold serial dilution of B. pseudomallei genomic DNA and B. pseudomallei suspension. Results & Discussion: The orf12 region of TTS1 gene cluster of B. pseudomallei LAMP assay showed high specificity towards B. pseudomallei strain. The orf12 LAMP assay of serially diluted B. pseudomallei genomic DNA gave a limit of detection (LOD) of 1.0 × 10-4 ng/μL while the LAMP assay of serially diluted B. pseudomallei suspension showed an LOD of 1.5 × 10-4 CFU/mL. Conclusion: High specificity and sensitivity of orf12 LAMP assay in this study proves that the orf12 region of TTS1 cluster gene in B. pseudomallei has the potential of being one of the DNA markers for rapid detection of melioidosis.

AB - Introduction: Burkholderia pseudomallei is a gram-negative motile bacterium responsible for melioidosis, an endemic human disease across much of Southeast Asia and northern Australia. Due to clinical manifestations that frequently 'mimic' other disease, a rapid diagnosis for septicemic and acute melioidosis patients is much needed. Serological tests such as IHA and ELISA have poor sensitivity due to high background in seropositivity in endemic area while PCR and RT-PCR require sophisticated equipments make it almost impossible for rapid detection in rural district. As a rapid, simple and cost effective detection method, LAMP assay give a better anticipation for early detection of melioidosis. Objective: To define a highly specific region in B. pseudomallei for assessment of a highly sensitive LAMP assay for rapid diagnosis of melioidosis. Methods: Specific marker for B. pseudomallei was determined based on analysis and alignment of conserved region of orf12 in TTS1 gene cluster amongst all B. pseudomallei strains and its distinction from other Burkholderiae. Primers were designed using Primer Explorer software and the primers specificity were validated using Burkholderiae and other Bacteriacea. The sensitivity of primers to amplify target region was determined by defining the limit of detection (LOD) of optimized LAMP assay on 10-fold serial dilution of B. pseudomallei genomic DNA and B. pseudomallei suspension. Results & Discussion: The orf12 region of TTS1 gene cluster of B. pseudomallei LAMP assay showed high specificity towards B. pseudomallei strain. The orf12 LAMP assay of serially diluted B. pseudomallei genomic DNA gave a limit of detection (LOD) of 1.0 × 10-4 ng/μL while the LAMP assay of serially diluted B. pseudomallei suspension showed an LOD of 1.5 × 10-4 CFU/mL. Conclusion: High specificity and sensitivity of orf12 LAMP assay in this study proves that the orf12 region of TTS1 cluster gene in B. pseudomallei has the potential of being one of the DNA markers for rapid detection of melioidosis.

UR - http://www.scopus.com/inward/record.url?scp=84896712681&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84896712681&partnerID=8YFLogxK

U2 - 10.1016/S2222-1808(14)60547-8

DO - 10.1016/S2222-1808(14)60547-8

M3 - Article

VL - 4

SP - 241

JO - Asian Pacific Journal of Tropical Disease

JF - Asian Pacific Journal of Tropical Disease

SN - 2222-1808

IS - 3

ER -