Establishment and application of dual rpa-basic and rpa-lfd detection method for pasteurella multocida and actinobacillus pleuropneumoniae
Based on recombinase polymerase amplification (RPA) detection technology (combined with (lateral flow dipstick, LFD)), it is aimed to establish a dual recombinase polymerase amplification method for the rapid identification of Pasteurella multocida (Pm) and Actinobacillus pleuropneumoniae (APP). The...
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| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Kafkas University, Faculty of Veterinary Medicine
2024-12-01
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| Series: | Kafkas Universitesi Veteriner Fakültesi Dergisi |
| Subjects: | |
| Online Access: | https://vetdergikafkas.org/pdf.php?id=3168 |
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| Summary: | Based on recombinase polymerase amplification (RPA) detection technology (combined
with (lateral flow dipstick, LFD)), it is aimed to establish a dual recombinase polymerase
amplification method for the rapid identification of Pasteurella multocida (Pm) and
Actinobacillus pleuropneumoniae (APP). The conserved fragments of Pm kmt1 gene
and APP ApxIV gene were selected for the amplification of target fragments. Eight
pairs of primers for Pm and APP, and one probe for KMT1Pn and APP323Pn were
designed. A single RPA-Basic primer screening test was performed. The reaction
time and temperature of double RPA were optimized. The optimal primers and probe
matching systems of dual RPA-LFD were explored. Dual RPA sensitivity and specificity
tests were performed. The method was used to detect 60 clinical samples. The results of
the primer screening test showed that the primers had the strongest specificity and the
highest amplification efficiency for ApxIV2698F/ApxIV3020R and KMT1F/KMT1R.
The method had the best amplification efficiency at a reaction temperature of 37ºC and a
reaction time of 35 min. The optimal primer ratio of KMT1F/KMT1R and ApxIV2698F/
ApxIV3020R was 2 μL : 1.5 μL, and the optimal probe ratio of KMT1Pn and APP323Pn
was 0.6 μL : 0.4 μL. The minimum detection limit of dual RPA-Basic and RPA-LFD
sensitivity test was 10-6 ng/μL. The specific test results showed no cross-reaction with
enteropathogenic Escherichia coli, Salmonella, Glaesserella parasuis, Staphylococcus
aureus, Streptococcus suis, Aeromonas hydrophila. Using 60 clinical samples of suspected
Pm and/or APP infection to evaluate the detection system, the detection rate of dual
RPA-Basic and RPA-LFD is higher than that of PCR, indicating that they have strong
practicability. This study successfully established a dual RPA-Basic and RPA-LFD
detection method for Pm and APP, which can be used for the rapid differential diagnosis
of Pm and APP mixed infection in clinical. |
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| ISSN: | 1309-2251 |