J Arthropod-Borne Dis, December 2018, 12(4): 432–433                                    PG de Mendonça: Troubleshooting Real-Time … 

432 

 http://jad.tums.ac.ir 

Published Online: December 25, 2018 
 

Letter to the Editor 

Troubleshooting Real-Time PCR Screening for Leishmania infantum 
 

Philippe Gil de Mendonça 
 

Institute of Comparative Tropical Medicine and Parasitology, Ludwig Maximilian University, Munich, 

Germany 
 

(Received 3 Jan 2018; accepted 13 Nov 2018) 

 
Dear Editor-in-Chief 
 

The parasite Leishmania infantum is a wide-

spread cause of concern to both human and vet-

erinary medicine (1-3). A simple and quick real-

time PCR protocol was proposed for screening 

samples for L. infantum (4). This protocol was 

efficiently applied in various studies (4-7). How-

ever, during informal discussions at internation-

al meetings, colleagues complained of difficul-

ties in applying this protocol. Such complaints 

are common and usually overcome through op-

timization, however, the opportunity to use a 

different type of thermocycler whilst on a visit 

abroad flagged the seriousness of that complaint.  

The reason for the difficulties encountered 

lies in a weakness of this PCR protocol. Indeed, 

the annealing temperature of the probe is sub-

stantially lower than the annealing temperature 

of the primers, whereas this should be the re-

verse. The implication of this is that if using a 

fast cooling thermocycler with Peltier element, 

the real-time PCR will work as expected (see 

Fig. 2 in reference 5), although the curves some-

times look somewhat "stretched", which is a 

symptom of suboptimal PCR conditions. How-

ever, if using a slow cooling (e.g. air-cooled) 

thermocycler, the real-time PCR may look like 

it failed, as no curves will appear. In such a case, 

the primers annealed much earlier than the probe, 

and the Taq polymerase was fast enough to elon-

gate the DNA target before the probe had at-

tached. This results in no fluorophore release 

and thus no positive signal detection. How-

ever, an agarose gel electrophoresis will reveal 

that the PCR amplification was successful, 

while the fluorogenic detection failed.  

 

 
This is an important limitation to be aware 

of. The implication is that researchers using a 

slow cooling thermocycler will have no chance 

to observe a real-time detection signal. They will 

have no other option than to run an agarose gel 

electrophoresis, thus losing both the specificity 

of the TaqMan probe and the sensitivity of the 

real-time detection. Researchers having a fast 

cooling thermocycler may continue using this 

relatively old protocol, whereas laboratories re-

lying on air-cooled equipment ought to opt for 

alternative real-time PCR protocols. Many real-

time PCR protocols (including TaqMan proto-

cols) have been developed and can be tested 

and selected to suit the technical requirements 

and limitations of each laboratory. Air-cooled, 

i.e. slow cooling thermocyclers, are however a 

limiting factor in many cases! If real-time de-

tection is unsuccessful, then sequencing of am-

plicons might be required to confirm specific 

amplification. Indeed, the added specificity of 

the TaqMan probe is missing in such cases. Re-

searchers in Asia (8) cautiously opted for se-

quencing their Leishmania isolates following 

conventional amplification. A wise example to 

follow is such cases indeed. 

 
Conflict of interest 
 
 

The author declare that there is no conflict 

of interest. 

 

Corresponding author: Dr Philippe Gil de Men-
donça, E-mail: pgm@cantab.net 



J Arthropod-Borne Dis, December 2018, 12(4): 432–433                                    PG de Mendonça: Troubleshooting Real-Time … 

433 

 http://jad.tums.ac.ir 

Published Online: December 25, 2018 
 

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