| Brucellosis is a worldwide re-emerging zoonosis,which has economic impact due to abortion and loss of fertility in different animal species.Rapid and sensitive diagnostic technique should be developed for accurate detection for prevention and control measures.In this study real time,recombinase polymerase amplification(RPA)targeting Brucella spp.bp26 gene was developed for detection of Brucella spp.in different samples of domestic animals.The analytical sensitivity of developed RPA is four copies per reaction and six colony form units(CFU)of bacteria bearing constructed plasmid with target sequence within 20 minutes at 40~oC reaction temperature.The diagnostic sensitivity of RPA is 94.9%and specificity 97.0%while the sensitivity of real time Polymerase Chain Reaction and Polymerase Chain Reaction were 91.5%and 93.2%and specificity are98.5%and 97.0%respectively.RPA showed sensitivity 62.2%compared to RBPT,which was 55.6%.There is no cross-reaction detected with Chlamydia abortus,Toxoplasma gondii and Salmonella typhimurium.In conclusion,the obtained results entitle RPA to be a field and point of care test of animal brucellosis.In this study,rapid lateral-flow dipstick combined with SYBR green I RPA has been developed to detect Brucellosis.The IS711 region belongs to bp 26 gene of Brucella spp.had been selected to design primers and probe.Different degrees of incubation temperature and incubation time have been tested for optimization and robustness.Lateral flow beside SYBR green I endpoint detection has been used.The developed and optimized RPA has validated for its analytical sensitivity and specificity.Diagnostic sensitivity of RPA was compared to Real-time PCR and PCR.The developed RPA can work in temperature between 30-35~oC within 10-30 minutes.The analytical sensitivity was 6 colony form units of bacteria-bearing plasmid and 6 copies of the plasmid.There is no cross-reactivity with Chlamydia abortus,Salmonella typhimurium,E.coli and Toxoplasma gondii.There is no significant difference between the positive results obtained by RPA,PCR,and qPCR in the screening of field samples which was 84.4%,86.6%,and 84.4%respectively.It is concluded that the Lateral flow combined with SYBR green I RPA can be a candidate to be used for diagnosis of Brucella spp.infection in the field and low capability laboratories.Duplex recombinase polymerase amplification for specific detection of B.melitensis and B.abortus has been developed.Specific sequence of B.melitensis hypothetical protein and B.abortus membrane transporter has been selected for primer design.Multiple sequence alignments were used for determination of specific regions of B.melitensis and B.abortus.The test was performed at 38~oC for 20 minutes incubation.The test was validated by determination of analytical sensitivity and specificity.The analytical sensitivity was determined by 10-fold serial dilution of constructed plasmids ligated to B.melitensis and B.abortus specific sequences.Analytical specificity was performed by screening DNA of B.melitensis bv.3,B.melitensis M5,B.abortus S19 and B.suis S2.Another non Brucella organism was tested including Chlamydia abortus,Salmonella typhimurium,E.coli and Toxoplasma gondii.Different field samples(n=62)were collected from Qinghai,Inner Mongolia and Xinjiang provinces.The field samples were screened by new developed Duplex RPA and multiplex AMOS PCR.The analytical sensitivity was 9×10~2 plasmid copies of Brucella melitensis and 9×10~1 plasmid copies of B.abortus.The test has specificity to B.melitensis and B.abortus and did not has cross reactivity to Chlamydia abortus,Salmonella typhimurium,E.coli and Toxoplasma gondii.Screening of field samples by RPA reveals high prevalence of B.melitensis in sheep and yak was 77.4%and low prevalence of B.abortus was 4.8%.The results of multiplex AMOS PCR showed the prevalence of B.melitensis was 19.3%and B.abortus was 4.8%.It concluded that the developed Duplex RPA is useful in epidemiological surveillances and clinical field. |
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