Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach

Trajano-Silva, Lays Adrianne Mendonça; Silva, Rômulo Pessoa e; Gonçalves-de-Albuquerque, Suênia da Cunha; Morais, Rayana Carla Silva de; Costa-Oliveira, Cíntia Nascimento da; Goes, Tayná Correia de; Paiva-Cavalcanti, Milena de

doi:10.1590/0037-8682-0012-2017

Cited by 5 publications

(2 citation statements)

References 29 publications

(56 reference statements)

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“…SYBR green due to its lower cost, but this method is prone to false–positives as it will visualise any amplified double-stranded DNA [ 178 ]. Use of probe-based real-time PCR increases specificity, thus avoids this issue, and targets can be multiplexed, whereby multiple assays occur in the same reaction, which saves reagents and time and increases throughput [ 179 ]. Real-time PCR was compared to cPCR, and higher sensitivity and specificity, 93.9% and 100%, respectively, for the former was reported compared to two cPCR assays tested (75.6% and 100% for kDNA, and 53.7% and 88.8% for ITS1, respectively) [ 180 ].…”

Section: Methods For the Detection And Diagnosis Of Leishmaniasismentioning

confidence: 99%

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

et al. 2022

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Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world’s poorest regions. Transmitted by the bite of a female sand fly, Leishmania infections are increasing with human movement (due to international travel and war) as well as with shifts in vector habitat (due to climate change). Accurate diagnosis of the 20 or so species of Leishmania that infect humans can lead to the successful treatment of infections and, importantly, their prevention through modelling and intervention programs. A multitude of laboratory techniques for the detection of Leishmania have been developed over the past few decades, and although many have drawbacks, several of them show promise, particularly molecular methods like polymerase chain reaction. This review provides an overview of the methods available to diagnostic laboratories, from traditional techniques to the now-preferred molecular techniques, with an emphasis on polymerase chain reaction-based detection and typing methods. Graphical abstract

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Section: Methods For the Detection And Diagnosis Of Leishmaniasismentioning

confidence: 99%

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

et al. 2022

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“…It is carried out via desiccation of the insect digestive system and microscopic examination, whose mean positivity rates range between 0 and 0.9%. However, this technique is hard to operationalize, and new strategies are required that might include molecular methods based on polymerase chain reaction (PCR), resulting in higher sensitivity and specificity [ 4 , 5 , 6 , 7 , 8 , 9 ]. In such a context, the objective of this study was to carry out L. infantum diagnosis in Lu.…”

Section: Introductionmentioning

confidence: 99%

Monitoring Leishmania infantum Infections in Female Lutzomyia longipalpis by Using DNA Extraction on Cation Exchange Paper and PCR Pool Testing

Coutinho

Marson

Rangel³

et al. 2022

Diagnostics

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Visceral leishmaniasis remains a serious public health issue, and Brazil was among the seven countries with the highest prevalence of this disease worldwide. The measures to control this disease are not easily developed, and the improvement of its diagnosis, surveillance, and control is still needed. This study aimed to carry out the polymerase chain reaction (PCR) diagnosis of Leishmania infantum in vector samples in some municipalities of the State of São Paulo, which included two municipalities with human disease transmission and two with dog transmission only. Vectors were collected in traps with luminous bait. Next, they were killed at −4 °C and kept in 70% alcohol. Groups of ten female insects (pools) were mashed on cation exchange paper (fine cellulose phosphate with 18 µEq/cm² ionic exchange capacity) for DNA extraction. The PCR was carried out to identify the natural infection of the Leishmania genus in female Lutzomyia longipalpis (Lu. Longipalpis). Out of the 3,880 Lu. longipalpis phlebotomines, 1060 were female and 2820 were male (3:1). The method used to extract the DNA in pools of ten phlebotomines and the PCR resulted in sensitivity, specificity, practicality, and faster analyses when compared to the individual analysis method. The procedure described can be used on a large scale in the leishmaniasis epidemiological surveillance, enabling a higher number of analyses and the optimization of human resources because the traditional diagnostic method is carried out via desiccation of the insect digestive system and microscopic examination, which is time-demanding and there is the need of manual skills.

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A rapid high-resolution melting method for differentiation of Leishmania species targeting lack gene

et al. 2018

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Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach

Abstract: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.

Cited by 5 publications

References 29 publications

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

Monitoring Leishmania infantum Infections in Female Lutzomyia longipalpis by Using DNA Extraction on Cation Exchange Paper and PCR Pool Testing

A rapid high-resolution melting method for differentiation of Leishmania species targeting lack gene

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