Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis

Abstract

BackgroundMolecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.ResultsTwo primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting the Leishmania braziliensis kinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously.PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD – 567 bp) as well as of small quantities (10 pg) of the target parasite’s DNA, detected by amplification of a 138 bp product.ConclusionsThe new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection of L. braziliensis kDNA.

Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)

“…Molecular methods employing polymerase chain reaction (PCR) based methods overcome these constraints along with provision of much higher sensitivity and specificity. Numerous PCR procedures [14–19] have been employed for diagnosis of leishmaniasis including triplex PCR [20], multiplex PCR [21], restriction fragment length polymorphism analysis and nested PCR [22]. Quantitative PCR (Q-PCR) is an exceptionally sensitive and specific assay for diagnosis of VL that enables rapid and accurate quantification of parasite burden [23].…”

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection

“…In last several years advances in the polymerase chain reaction (PCR) based methods have been introduced for diagnosis of leishmaniasis [12–15]. Several PCR protocols have been developed for detection of Leishmania parasite, such as triplex PCR [16], multiplex PCR [17], restriction fragment length polymorphism (RFLP) analysis and nested PCR [18]. Quantitative Real-time PCR (Q-PCR) has emerged as a highly sensitive tool for VL and PKDL diagnosis with simultaneous quantification of the parasite burden [19–22].…”

Leishmaniases diagnosis: an update on the use of immunological and molecular tools

“…Mohammadiha et al [ 41 ] performed this multiplex format for humans and dogs, using TaqMan-based qPCR, having reached a very good sensitivity in both cases (93.9–100 %, respectively). Gonçalves-de-Albuquerque et al [ 64 ] standardized a triplex PCR to CL capable to monitor not only the sample quality, but also small losses of DNA during the extraction process by using a plasmid as reporter.…”