Abstract
ObjectiveThe performance of comparative analysis of sensitivity and resultsof detection of avian influenza virus by real time polymerase chainreaction (PCR-RT) and loop-mediated isothermal amplification of thenucleic acids (LAMP) was the main goal of the study.IntroductionAs part of this surveillance study for Avian Influenza both activeand passive surveillance samples were tested using PCR and alsoutilized to validate the LAMP method. Active surveillance samplesinclude pathological material and tracheal and cloacal swabs fromill poultry, which were subsequently assessed for avian influenzaduring diagnosis, and birds collected by hunters. Passive surveillanceincluded environmental samples such as sand and bird faeces.Active surveillance samples were taken mostly from poultry farmsacross Ukraine, where infected birds are required to be diagnosedby State Scientific Research Institute of Laboratory Diagnosticsand Veterinary Sanitary Expertise (SSRILDVSE) by Ukraine Law.Passive surveillance samples were taken primarily during the annualbird migration season. Development of simple, sensitive, and cheapmethods for diagnostics of avian influenza is a very important taskfor practical veterinary medicine. LAMP is one of such methods.The technique is based on isothermal amplification of nucleic acids.It does not require special conditions and equipment (PCR cyclers),therefore it is cheaper in comparison with PCR. Accurate diagnosisis necessary for determining the risk associated with avian influenzain Ukraine and along the Dnipro River during the migratory season.MethodsFor the research, we used PCR-RT commercial kit Bird-Flu-PCR(Ukrzoovetprompostach, Ukraine), LAMP (the protocol has beenoptimized and patented by SSRILDVSE), QIAamp®Viral RNA MiniKit. For the study, we used pathological and biological materials frombirds, which were sent to the SSRILDVSE from all regions of Ukraineaccording to the 2013–2014 State monitoring plan.Set up of the real time PCR reactions and parameters ofamplifications are indicated in the instruction to the kit.The following protocol was used to set up the RT- LAMP: 2.5μL10 X Thermopol buffer, 1 mmol/L betaine, 5 mmol/L MgSO4,1.4 mmol/L - BNTP, 12.5μmol/L SYBR GREEN, 0.5 mmol/LMnCL2, up to 25μL Nuclease-free water, 8 U Bsm DNA polymerase,0.1μM/1 of F3, 0.1μM/1 of B3, 0.8μM/1 of FIP, 0.8μM/1 of BIP,0.4μM/1 of LF, 0.4 of LB, 2μL cDNA.During our work, we used the following optimal temperature andtime for the amplification – 59°C and 60 minutes.The sensitivity of diagnostic kit Bird-Flu-PCR and RT- LAMP wasdetermined by testing cDNA of the reference strain of AIV H5N1,which was provided to us by NSC Institute for Experimental andClinical Veterinary Medicine (Kharkiv, Ukraine). For the standard,we employed concentration in the range of 10.0-0.01 ng/sample.ResultsTable 1.This table shows the reproducibility results obtained by bothmethods. However, taken into account absence of highly pathogenicavian influenza virus circulating in Ukraine during the studied period,it was not possible to confirm these results with protocols of positivesamples.Table 2.It has been established that the sensitivity of PCR-RT kit Bird-Flu-PCR is 0.01 ng/sample for gene M and 0.1 ng/sample for subtypeH5N1.Fig. 1. Visual detection of LAMP products with differentconcentrations of cDNA of avian influenza virus (ng per sample):1 – 10; 2 – 5; 3 – 1.0; 4 – 0.1; 5–7 – 0.01; 8–9 – 0.1; 10 – negative.We have examined the LAMP results using electrophoresis forthe confirmation of visual detection and correct interpretation of theresults (Fig. 2).Fig.2. Electrophoresis results for LAMP products. M –molecular weight marker; 1 – 10.0; 2 – 5.0; 3 – 1.0; 4 – 0.1; 5–7– 0.01; 8 - negative control.It has been established that the sensitivity of LAMP is0.1 ng/sample. Slightly lower sensitivity of LAMP in comparisonto PCR-RT can be explained by visual detection of the products ofthe LAMP reaction.Conclusions1. Sensitivity of both methods is high.2. LAMP is a perspective screening method for the diagnosis ofviral infectious diseases supported by confirmation of positive resultsby PCR-RT.