Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

Myrthe Pareyn, Rik Hendrickx, Nigatu Girma, Sarah Hendrickx, Lieselotte Van Bockstal, Natalie Van Houtte, Simon Shibru, Louis Maes, Herwig Leirs, Guy Caljon

Research output: Contribution to journalA1: Web of Science-articlepeer-review

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Abstract

Background: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.

Methods: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.

Results: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (Delta Cq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10(-3) and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.

Conclusions: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Original languageEnglish
Article number276
JournalParasites and Vectors
Volume13
Issue number1
Number of pages10
ISSN1756-3305
DOIs
Publication statusPublished - 1-Jun-2020

Keywords

  • Spliced leader RNA
  • Kinetoplast DNA
  • Nucleic acid extraction
  • Sand fly
  • Reservoir
  • Real-time PCR
  • REAL-TIME PCR
  • CUTANEOUS LEISHMANIASIS
  • DNA
  • QUANTIFICATION
  • INFECTION
  • DIAGNOSIS
  • TROPICA

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