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Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis.
Diagn Microbiol Infect Dis. 2015 Jul; 82(3):199-200.DM

Abstract

Early diagnosis of leptospirosis in humans is critical with regard to initiation of appropriate treatment; however, the gold standard serological test cannot detect antibodies until nearly a week after symptom onset. PCR has been shown to be sensitive and specific in the early phase of leptospirosis. Previously, we developed and validated a TaqMan PCR assay targeting lipL32. We reoptimized and validated this assay using PerfeCTa® qPCR ToughMix®, Low ROX™ (Quanta Biosciences, Gaithersburg, MD, USA). For optimization with the new mix, the final primer concentrations were increased from 0.5 μmol/L to 0.9 μmol/L compared to our previous assay, and the probe concentration increased from 0.1 μmol/L to 0.125 μmol/L. This newly optimized assay resulted in a lower limit of detection and increased diagnostic sensitivity. Here, we present the performance data of the improved assay and describe several clinical cases that were initially negative but tested positive using the optimized assay.

Authors+Show Affiliations

Bacterial Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA, USA. Electronic address: rgalloway@cdc.gov.Bacterial Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA, USA.

Pub Type(s)

Comparative Study
Journal Article
Validation Study

Language

eng

PubMed ID

25912810

Citation

Galloway, Renee L., and Alex R. Hoffmaster. "Optimization of LipL32 PCR Assay for Increased Sensitivity in Diagnosing Leptospirosis." Diagnostic Microbiology and Infectious Disease, vol. 82, no. 3, 2015, pp. 199-200.
Galloway RL, Hoffmaster AR. Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis. Diagn Microbiol Infect Dis. 2015;82(3):199-200.
Galloway, R. L., & Hoffmaster, A. R. (2015). Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis. Diagnostic Microbiology and Infectious Disease, 82(3), 199-200. https://doi.org/10.1016/j.diagmicrobio.2015.03.024
Galloway RL, Hoffmaster AR. Optimization of LipL32 PCR Assay for Increased Sensitivity in Diagnosing Leptospirosis. Diagn Microbiol Infect Dis. 2015;82(3):199-200. PubMed PMID: 25912810.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis. AU - Galloway,Renee L, AU - Hoffmaster,Alex R, Y1 - 2015/04/09/ PY - 2015/02/12/received PY - 2015/03/13/revised PY - 2015/03/16/accepted PY - 2015/4/28/entrez PY - 2015/4/29/pubmed PY - 2016/2/26/medline KW - Diagnosis KW - Leptospirosis KW - Real time PCR SP - 199 EP - 200 JF - Diagnostic microbiology and infectious disease JO - Diagn. Microbiol. Infect. Dis. VL - 82 IS - 3 N2 - Early diagnosis of leptospirosis in humans is critical with regard to initiation of appropriate treatment; however, the gold standard serological test cannot detect antibodies until nearly a week after symptom onset. PCR has been shown to be sensitive and specific in the early phase of leptospirosis. Previously, we developed and validated a TaqMan PCR assay targeting lipL32. We reoptimized and validated this assay using PerfeCTa® qPCR ToughMix®, Low ROX™ (Quanta Biosciences, Gaithersburg, MD, USA). For optimization with the new mix, the final primer concentrations were increased from 0.5 μmol/L to 0.9 μmol/L compared to our previous assay, and the probe concentration increased from 0.1 μmol/L to 0.125 μmol/L. This newly optimized assay resulted in a lower limit of detection and increased diagnostic sensitivity. Here, we present the performance data of the improved assay and describe several clinical cases that were initially negative but tested positive using the optimized assay. SN - 1879-0070 UR - https://www.unboundmedicine.com/medline/citation/25912810/full_citation L2 - https://linkinghub.elsevier.com/retrieve/pii/S0732-8893(15)00116-9 DB - PRIME DP - Unbound Medicine ER -