TaqMan real-time PCR for detection of pathogenic  spp. in canine clinical samples

Target gene	Nucleotide sequence	Reference
LipL32(1)
Forward	5′-AAG CAT TAC CGC TTG TGG TG-3′	36
Reverse	5′-GAA CTC CCA TTT CAG CGA TT-3′	36
TaqMan probe	FAM/ZEN-5’-AA AGC CAG GAC AAG CGC CG-3’-IBFQ
LipL32(2)
Forward	5′-CGG GAG GCA GCA GTT AAG AAT-3′	41
Reverse	5′-ACG TAT GGT GCA AGC GTT GTT-3′	41
TaqMan probe	FAM/ZEN-5′-GCA ATG TGA TGA TGG TAC CTG CCT-3′-IBFQ

Threshold cycle (Ct) values of fivefold serial dilution of Leptospira genomic DNA in a 20 μL reaction volume

Dilution factor	Mean DNA copy number/μL	log DNA copies	Mean Ct value
Standard control with DNA concentration of 51.85 ng/μL (primary stock)	10,382,320	7.01629441	12.47
1:5	2,076,464	6.317324406	14.15
1:25	415,292.8	5.618354402	17.66
1:125	83,058.56	4.919384397	18.96
1:625	16,611.712	4.220414393	21.05
1:3,125	3,322.3424	3.521444389	23.36
1:15,625	664.46848	2.822474384	25.79
1:78,125	132.893696	2.12350438	27.43
1:390,625	26.5787392	1.424534376	29.67
1:1,953,125	5.31574784	0.725564371	32.00

Comparison of Ct values between a qPCR assay with the LipL32(1) primers and one with the LipL32(2) primers

Group	DNA concentration (ng/μL)	LipL32(1) Ct value	LipL32(2) Ct value
Positive control
L. borgpetersenii serovar Ballum strain Mus 127	26.4	15.33	32.08
L. borgpetersenii serovar Hardjobovis strain 117123	20.6	17.41	33.36
L. borgpetersenii serovar Javanica*	8.2	16.22	36.14
L. borgpetersenii serovar Javanica strain Veldrat Bataviae 46	41.0	14.35	33.96
L. borgpetersenii serovar Tarassovi strain Perepelitsin	16.1	20.54	36.39
L. interrogans serovar Australis*	9.9–11.9	15.43–15.58	33.78–34.38
L. interrogans serovar Australis strain Ballico	11.8	29.67	N
L. interrogans serovar Autumnalis strain Akiyami A	32.6	23.56	N
L. interrogans serovar Bataviae*	5.3–20.1	13.91–16.02	34.11–35.91
L. interrogans serovar Bataviae strain Swart	15.8	17.97	34.29
L. interrogans serovar Canicola strain Hond Utrecht IV	12.7	14.99	34.08
L. interrogans serovar Copenhageni strain M20	2.5	17.95	34.44
L. interrogans serovar Djasiman strain Djasiman	11.5	16.20	35.43
L. interrogans serovar Hebdomadis strain Hebdomadis	28.4	16.77	33.91
L. interrogans serovar Icterohaemorrhagiae strain RGA	16.8	14.3	32.81
L. interrogans serovar Lai strain Lai	12.8	19.22	36.19
L. interrogans serovar Pomona strain Pomona	7.2	18.05	35.44
L. interrogans serovar Pyrogenes strain Salinem	43.3	21.40	N
L. kirschneri serovar Cynopteri strain 3522C	29.7	14.44	33.60
L. kirschneri serovar Grippotyphosa strain Moskva V	42.3	13.04	31.79
L. kmetyi serovar Malaysia strain Bejo-ISO9	36.1	18.78	33.34
L. weilii serovar Celledoni strain Celledoni	13.0	24.15	40.52
Non-target negative control
Bacillus spp.	9.9	N	N
Escherichia coli	16.0	N	N
L. biflexa serovar Patoc strain Patoc I	42.9	N	N
L. selangorensis**	8.7-14.3	N	N
L. wolffii**	6.7-19.3	N	N
Pseudomonas aeruginosa	84.9	N	N
Salmonella spp.	41.1	N	N
Staphylococcus aureus	14.4	N	N
Streptococcus spp.	4.2	N	N
No-target negative control
RNase-free water	0	N	N

List of bacteria used in the qPCR assay

Bacterium (n = 44)	Number tested (n)	Source
Pathogenic group
L. borgpetersenii serovar Ballum strain Mus 127	1	FVM, UPM
L. borgpetersenii serovar Hardjobovis strain 117123	1	FVM, UPM
L. borgpetersenii serovar Javanica	1	Dog
L. borgpetersenii serovar Javanica strain Veldrat Bataviae 46	1	FVM, UPM
L. borgpetersenii serovar Tarassovi strain Perepelitsin	1	FVM, UPM
L. interrogans serovar Australis	2	Dog
L. interrogans serovar Australis strain Ballico	1	FVM, UPM
L. interrogans serovar Autumnalis strain Akiyami A	1	FVM, UPM
L. interrogans serovar Bataviae	8	Dog
L. interrogans serovar Bataviae strain Swart	1	FVM, UPM
L. interrogans serovar Canicola strain Hond Utrecht IV	1	FVM, UPM
L. interrogans serovar Copenhageni strain M20	1	FVM, UPM
L. interrogans serovar Djasiman strain Djasiman	1	FVM, UPM
L. interrogans serovar Hebdomadis strain Hebdomadis	1	FVM, UPM
L. interrogans serovar Icterohaemorrhagiae strain RGA	1	FVM, UPM
L. interrogans serovar Lai strain Lai	1	FVM, UPM
L. interrogans serovar Pomona strain Pomona	1	FVM, UPM
L. interrogans serovar Pyrogenes strain Salinem	1	FVM, UPM
L. kirschneri serovar Cynopteri strain 3522C	1	FVM, UPM
L. kirschneri serovar Grippotyphosa strain Moskva V	1	FVM, UPM
L. kmetyi serovar Malaysia strain Bejo-ISO9	1	FVM, UPM
L. weilii serovar Celledoni strain Celledoni	1	FVM, UPM
Intermediate group
L. selangorensis	3	Environmental
L. wolffii	5	Environmental
Saprophytic group
L. biflexa serovar Patoc strain Patoc I	1	FVM, UPM
Other bacteria
Bacillus spp. (Gram positive)	1	Clinical
Escherichia coli (Gram negative)	1	Clinical
Pseudomonas aeruginosa (Gram negative)	1	Clinical
Salmonella spp. (Gram negative)	1	Clinical
Staphylococcus aureus (Gram positive)	1	Clinical
Streptococcus spp. (Gram positive)	1	Clinical

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TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples

Publicado en línea: 16 jun 2023

Páginas: 187 - 195

Recibido: 27 oct 2022

Aceptado: 18 abr 2023

DOI: https://doi.org/10.2478/jvetres-2023-0024

Palabras clavecanine leptospirosis, qPCR, sensitivity, specificity

© 2023 Mohammad Sabri Abdul Rahman et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Primers and TaqMan probes for qPCR of pathogenic Leptospira spp.

Threshold cycle (Ct) values of fivefold serial dilution of Leptospira genomic DNA in a 20 μL reaction volume

Comparison of Ct values between a qPCR assay with the LipL32(1) primers and one with the LipL32(2) primers

List of bacteria used in the qPCR assay

Palabras clave
canine leptospirosis, qPCR, sensitivity, specificity