@article{Chagas_Vaucher_Brandelli_2018, title={Detection of Paenibacillus larvae by Real-Time PCR}, volume={38}, url={https://seer.ufrgs.br/index.php/ActaScientiaeVeterinariae/article/view/17058}, DOI={10.22456/1679-9216.17058}, abstractNote={<strong><em><span style="font-size: x-small;"><span style="font-size: x-small;"><strong><em><font size="2"><font size="2"><p align="left"><span style="font-family: Times New Roman;">Background</span></p></font></font><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">: </span></span></span></em></strong></span><strong><em><font size="2"><p align="left"> </p></font><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">: </span></span></span></em></strong></span><p align="left"><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">: </span></span></span></p></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times New Roman;">is the agent of the American Foulbrood disease (AFB), which may determine the death of the </span>hive. The detection strategy for its diagnosis is based on clinical signs of disease, isolation and identification of </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">P. larvae</span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">, which usually employs microbiological and biochemical methods. Recently, molecular methods based on analysis of 16S rDNA by conventional PCR have been adopted, providing greater security and analytical speed. The rapid diagnosis is important to minimize economic losses and assess routes of spread of the pathogen. Despite the strong existing sanitary control, </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">P. larvae </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">was recently identified in the Brazilian states of Rio Grande do Sul and Paraná. After that, outbreaks have been reported in neighboring countries. This investigation was conducted to develop a protocol for detection of </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">P. larvae </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">by real-time PCR, allowing the reduction in the time of diagnosis, without loss of robustness found in the conventional PCR methods. </span></span><strong><em><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">Materials, Methods & Results</span></span></span><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">: </span></span></span></em><span style="font-family: Times New Roman;"><span style="font-size: x-small;"><span style="font-size: x-small;">Twenty-nine (29) </span></span><em><span style="font-size: x-small;"><span style="font-size: x-small;">P. larvae </span></span></em></span><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">strains were evaluated by real-time PCR using SYBR Green. The primers </span></span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">Pltr</span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">-F/R were designed according to the sequence X60619 of 16S rDNA gene published in GenBank, to amplify a fragment of 74 base pairs. The target gene is highly conserved and specific to </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">P. larvae. </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">The amplification conditions consisted of 1 cycle of 50°C for 2 minutes and 1 cycle of 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The fluorescence was monitored during the annealing at 60°C. The reactions were conducted in a 7500 Real Time PCR System equipment, using SYBRGreen PCR master mix (both Applied Biosystems), containing 2X Platinum SYBRGreen qPCR Supermix-UDG. The concentrations of primers were 1, 10 and 100 mM, and different concentrations of MgCl</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">2 </span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">(0,0mM de MgCl</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">2, </span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">1.0mM de MgCl</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">2 , </span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">2.0mM de MgCl</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">2 </span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">and 3.0mM de MgCl</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">2</span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">) were tested, with a final volume of 50mL; 25mL and 15mL, containing a 5mL sample. The analysis of the </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">melting </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">curve was made based on a 95°C for 15 seconds and 60°C for 20 seconds and one cycle with temperature ranging between 60°C and 95°C for 20 minutes. The best results of sensitivity and specificity in the reaction with SYBR Green were obtained with primer concentration set as 100 mM. The different concentrations of MgCl</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">2 </span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">tested did not affect the performance of the reaction. No amplification was observed with DNA obtained from </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">Paenibacillus alvei </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">or </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">Bacillus </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">species. The limit of detection was set as 6 pg of DNA template. The regression analysis of the C</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">T </span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">values of the PCR products showed a linear relationship between the initial amounts of DNA template and the values of C</span></span><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">T </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">(R</span></span></em><span style="font-family: Times-Roman; font-size: xx-small;"><span style="font-family: Times-Roman; font-size: xx-small;">2 </span></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">= 0.9982), indicating that the test is highly precise. </span></span><strong><em><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">Discussion</span></span></span><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">: </span></span></span></em><span style="font-family: Times New Roman;"><span style="font-size: x-small;"><span style="font-size: x-small;">The protocol developed allowed the unequivocal identification of </span></span><em><span style="font-size: x-small;"><span style="font-size: x-small;">P. larvae, </span></span></em></span><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times New Roman;">as all strains were detected by this </span>approach. The amplification of the expected 16S rDNA gene fragment was verified by amplification with the primers </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">Pltr </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">F/R only for chromosomal DNA of </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">P. larvae. </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">In addition, the amplicon specificity was verified by sequencing and no amplification was observed when the primers were tested with DNA from other bacterial species. The protocol developed in this study proved to be sensitive and specific, providing a rapid and accurate diagnostic tool. The results showed that the analysis by real-time PCR of partial 16S rDNA gene of </span></span><em><span style="font-family: Times-Italic; font-size: x-small;"><span style="font-family: Times-Italic; font-size: x-small;">P. larvae </span></span></em><span style="font-family: Times-Roman; font-size: x-small;"><span style="font-family: Times-Roman; font-size: x-small;">represents an important alternative for rapid diagnosis of AFB disease. The use of this methodology may represent an advance for rapid confirmation of the presence of this bacterium, what will allow the adoption of control measures against AFB, which can avoid its spreading in Brazilian territory.</span></span></strong></strong></strong><span style="font-size: x-small;"><span style="font-size: x-small;"><span style="font-family: Times New Roman;">Paenibacillus larvae </span></span></span>}, number={3}, journal={Acta Scientiae Veterinariae}, author={Chagas, Sérgio Salla and Vaucher, Rodrigo Almeida and Brandelli, Adriano}, year={2018}, month={Jun.}, pages={251–256} }