Development and validation of pan- Leptospira Taqman qPCR for the detection of Leptospira spp. in clinical specimens

Ali, Mohammad Ridhuan Mohd; Safee, Amira Wahida Mohd; Ismail, Nor Hayati; Sapian, Roslinda Abu; Hussin, Hani Mat; İsmail, Nabilah; Yean, Chan Yean

doi:10.1016/j.mcp.2018.03.001

Cited by 11 publications

(6 citation statements)

References 27 publications

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“…Analytically, the LipL32(1) assay amplified as few as approximately five DNA copies per reaction, which was close to the lowest theoretically possible LOD reported of three copies per reaction (7). The LipL32(1) assay LOD was comparable to previous molecular assays, which amplified between 1 and 10 copies per reaction (4,5,27,29,39). In terms of leptospiral coverage, another published assay that utilised a similar target showed the same results: it amplified all the pathogenic Leptospira spp.…”

Section: Discussionsupporting

confidence: 67%

“…Routinely, conventional PCR has been adopted to diagnose canine leptospirosis locally (32). To date, several qPCR methods have been described in human leptospirosis, and the superior usefulness of the qPCR as a diagnostic tool was demonstrated over the conventional PCR (8,29,39), but the validity of this technique has not been determined. To our knowledge, this study was the first to evaluate the analytical performance of a TaqMan probe-based qPCR to detect Leptospira spp.…”

Section: Discussionmentioning

confidence: 99%

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TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples

Rahman

Khor

Khairani-Bejo³

et al. 2023

Journal of Veterinary Research

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Introduction Canine leptospirosis has always been a differential diagnosis in dogs presenting with clinical signs and blood profiles associated with kidney and/or liver disease. The conventional polymerase chain reaction (PCR) provides diagnoses, but real-time PCR-based tests provide earlier confirmation and determine the severity of infection, especially in the acute stage, allowing early detection for immediate treatment decisions. To our knowledge, real-time PCR has not been routinely adopted for clinical investigation in Malaysia. This study evaluated TaqMan real-time PCR (qPCR) assays diagnosing leptospirosis and compared their applicability to clinical samples from dogs with kidney and/or liver disease against a conventional PCR reference. Material and Methods The qPCR assays were validated using existing leptospiral isolates. Whole blood and urine samples were analysed using a conventional PCR, LipL32(1) and LipL32(2) qPCRs and a microscopic agglutination test. The sensitivity and specificity of the qPCRs were determined. Results The LipL32(1) qPCR assay had more diagnostic value than the LipL32(2) qPCR assay. Further evaluation of this assay revealed that it could detect as low as five DNA copies per reaction with high specificity for the tested leptospiral strains. No cross-amplification was observed with other organisms. Analysing the clinical samples, the LipL32(1) qPCR assay had 100.0% sensitivity and >75.0% specificity. Conclusion The LipL32(1) qPCR assay is sensitive, specific and has the potential to be applied in future studies.

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples

Rahman

Khor

Khairani-Bejo³

et al. 2023

Journal of Veterinary Research

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“…qPCR detection of Leptospira DNA was carried out, following a previous protocol [ 5 ]. Briefly, following DNA extraction, 8 µL of patient DNA was added to a PCR mix containing 1× Biorad SsoAdvanced™ Universal Probes Supermix, 200 nM forward and reverse primers, 100 nM probe, and PCR-grade water (adjusted to a total volume of 20 µL).…”

Section: Methodsmentioning

confidence: 99%

Putative Pathogenic Genes of Leptospira interrogans and Leptospira weilii Isolated from Patients with Acute Febrile Illness

et al. 2022

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Leptospirosis is an important worldwide tropical disease caused by pathogenic Leptospira spp. The determination of virulence genes is important, as it influences patients’ clinical manifestations and clinical outcomes. This case report focused on detecting the pathogenic genes of Leptospira in association with the clinical manifestations of patients at the Hospital Universiti Sains Malaysia, Malaysia, who presented with acute febrile illness. Two cases were found and, to the best of our knowledge, these were the first two cases in Malaysia in which patients presented with febrile illness were associated with successful Leptospira isolation from clinical samples. Both clinical isolates were identified by 16S rRNA sequencing as Leptospira weilii and Leptospira interrogans, respectively, and they were classified as pathogenic Leptospira by the presence of different pathogenic genes, based on a polymerase chain reaction (PCR) amplification of targeted genes. This report emphasizes that different infecting Leptospira species and the presence of different virulence factors cause a slight difference in clinical manifestations and laboratory findings of leptospirosis. Genomic sequencing and annotation revealed the detection of classical leptospiral virulence factor genes that were otherwise missed using PCR for detection of Leptospira weilii genome B208.

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“…In addition, quantitative PCR provided the most reliable diagnosis result of Leptospira and qPCR showed high reliability, sensitivity, and specificity. Many previous studies have developed and validated qPCR tests for specific genes, such as the rrs gene [ 104 ], lipL32 gene [ 105 ], and lfb1 [ 106 ]. However, there are still many problems to be overcome when using qPCR to detect Leptospira including the need for stable DNA extraction technology, technical expertise, and expensive instruments are required.…”

Section: The Diagnosis Of Leptospiramentioning

confidence: 99%

Insight into the Structure, Functions, and Dynamics of the Leptospira Outer Membrane Proteins with the Pathogenicity

Hsu

Yang

2022

Membranes

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Leptospirosis is a widespread zoonosis that frequently occurs in tropical and subtropical countries. Leptospira enters the host through wounds or mucous membranes and spreads to the whole body through the blood, causing systemic infection. Kidneys are the preferential site where Leptospira accumulates, especially in the renal interstitium and renal tubule epithelial cells. Clinical symptoms in humans include high fever, jaundice, renal failure, and severe multiple-organ failure (Weil’s syndrome). Surface-exposed antigens are located at the outermost layer of Leptospira and these potential virulence factors are likely involved in primary host-pathogen interactions, adhesion, and/or invasion. Using the knockout/knockdown techniques to the evaluation of pathogenicity in the virulence factor are the most direct and effective methods and many virulence factors are evaluated including lipopolysaccharides (LPS), Leptospira lipoprotein 32 (LipL32), Leptospira ompA domain protein 22 (Loa22), LipL41, LipL71, Leptospira immunoglobulin-like repeat A (LigA), LigB, and LipL21. In this review, we will discuss the structure, functions, and dynamics of these virulence factors and the roles of these virulence factors in Leptospira pathogenicity. In addition, a protein family with special Leucine-rich repeat (LRR) will also be discussed for their vital role in Leptospira pathogenicity. Finally, these surface-exposed antigens are discussed in the application of the diagnosis target for leptospirosis and compared with the serum microscope agglutination test (MAT), the gold standard for leptospirosis.

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Development and validation of pan- Leptospira Taqman qPCR for the detection of Leptospira spp. in clinical specimens

Cited by 11 publications

References 27 publications

TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples

TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples

Putative Pathogenic Genes of Leptospira interrogans and Leptospira weilii Isolated from Patients with Acute Febrile Illness

Insight into the Structure, Functions, and Dynamics of the Leptospira Outer Membrane Proteins with the Pathogenicity

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