TY - JOUR AU - Sapachova, Maryna PY - 2017/05/02 Y2 - 2024/03/29 TI - Comparative Analysis of Methods of Molecular Detection of Avian Influenza Virus JF - Online Journal of Public Health Informatics JA - OJPHI VL - 9 IS - 1 SE - One Health DO - 10.5210/ojphi.v9i1.7739 UR - https://ojphi.org/ojs/index.php/ojphi/article/view/7739 SP - AB - <div style="left: 79.8182px; top: 297.773px; font-size: 12.564px; font-family: sans-serif; transform: scaleX(1.08117);" data-canvas-width="56.563006734006734">Objective</div><div style="left: 93.1212px; top: 311.033px; font-size: 12.564px; font-family: serif; transform: scaleX(0.995456);" data-canvas-width="335.5183878787879">The performance of comparative analysis of sensitivity and results</div><div style="left: 79.8182px; top: 325.814px; font-size: 12.564px; font-family: serif; transform: scaleX(1.03336);" data-canvas-width="349.8626759259258">of detection of avian influenza virus by real time polymerase chain</div><div style="left: 79.8182px; top: 340.595px; font-size: 12.564px; font-family: serif; transform: scaleX(0.987581);" data-canvas-width="348.3034868686869">reaction (PCR-RT) and loop-mediated isothermal amplification of the</div><div style="left: 79.8182px; top: 355.376px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00363);" data-canvas-width="271.4823299663299">nucleic acids (LAMP) was the main goal of the study.</div><div style="left: 79.8182px; top: 383.504px; font-size: 12.564px; font-family: sans-serif; transform: scaleX(1.11744);" data-canvas-width="73.28565488215487">Introduction</div><div style="left: 93.1212px; top: 396.764px; font-size: 12.564px; font-family: serif; transform: scaleX(1.02576);" data-canvas-width="336.3463537037037">As part of this surveillance study for Avian Influenza both active</div><div style="left: 79.8182px; top: 411.545px; font-size: 12.564px; font-family: serif; transform: scaleX(0.96756);" data-canvas-width="350.2081851851851">and passive surveillance samples were tested using PCR and also</div><div style="left: 79.8182px; top: 426.326px; font-size: 12.564px; font-family: serif; transform: scaleX(1.02665);" data-canvas-width="349.9330341750844">utilized to validate the LAMP method. Active surveillance samples</div><div style="left: 79.8182px; top: 441.107px; font-size: 12.564px; font-family: serif; transform: scaleX(0.964272);" data-canvas-width="350.255928282828">include pathological material and tracheal and cloacal swabs from</div><div style="left: 79.8182px; top: 455.888px; font-size: 12.564px; font-family: serif; transform: scaleX(0.982558);" data-canvas-width="350.4456442760942">ill poultry, which were subsequently assessed for avian influenza</div><div style="left: 79.8182px; top: 470.669px; font-size: 12.564px; font-family: serif; transform: scaleX(0.993468);" data-canvas-width="348.75704629629627">during diagnosis, and birds collected by hunters. Passive surveillance</div><div style="left: 79.8182px; top: 485.451px; font-size: 12.564px; font-family: serif; transform: scaleX(1.02942);" data-canvas-width="352.0952939393938">included environmental samples such as sand and bird faeces.</div><div style="left: 79.8182px; top: 500.232px; font-size: 12.564px; font-family: serif; transform: scaleX(1.03519);" data-canvas-width="349.72070303030296">Active surveillance samples were taken mostly from poultry farms</div><div style="left: 79.8182px; top: 515.013px; font-size: 12.564px; font-family: serif; transform: scaleX(0.963311);" data-canvas-width="350.34010690235675">across Ukraine, where infected birds are required to be diagnosed</div><div style="left: 79.8182px; top: 529.794px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00905);" data-canvas-width="351.06379175084203">by State Scientific Research Institute of Laboratory Diagnostics</div><div style="left: 79.8182px; top: 544.575px; font-size: 12.564px; font-family: serif; transform: scaleX(1.03548);" data-canvas-width="349.6804983164982">and Veterinary Sanitary Expertise (SSRILDVSE) by Ukraine Law.</div><div style="left: 79.8182px; top: 559.356px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00707);" data-canvas-width="349.26965639730633">Passive surveillance samples were taken primarily during the annual</div><div style="left: 79.8182px; top: 574.137px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01968);" data-canvas-width="349.75085656565653">bird migration season. Development of simple, sensitive, and cheap</div><div style="left: 79.8182px; top: 588.919px; font-size: 12.564px; font-family: serif; transform: scaleX(1.0381);" data-canvas-width="349.9945976430975">methods for diagnostics of avian influenza is a very important task</div><div style="left: 79.8182px; top: 603.7px; font-size: 12.564px; font-family: serif; transform: scaleX(0.970541);" data-canvas-width="350.62405269360255">for practical veterinary medicine. LAMP is one of such methods.</div><div style="left: 79.8182px; top: 618.481px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01584);" data-canvas-width="349.464397979798">The technique is based on isothermal amplification of nucleic acids.</div><div style="left: 79.8182px; top: 633.262px; font-size: 12.564px; font-family: serif; transform: scaleX(1.02052);" data-canvas-width="349.68049831649824">It does not require special conditions and equipment (PCR cyclers),</div><div style="left: 79.8182px; top: 648.043px; font-size: 12.564px; font-family: serif; transform: scaleX(1.02511);" data-canvas-width="349.83629158249124">therefore it is cheaper in comparison with PCR. Accurate diagnosis</div><div style="left: 79.8182px; top: 662.824px; font-size: 12.564px; font-family: serif; transform: scaleX(1.0124);" data-canvas-width="349.37896296296293">is necessary for determining the risk associated with avian influenza</div><div style="left: 79.8182px; top: 677.605px; font-size: 12.564px; font-family: serif; transform: scaleX(1.0008);" data-canvas-width="340.9234090909089">in Ukraine and along the Dnipro River during the migratory season.</div><div style="left: 79.8182px; top: 705.733px; font-size: 12.564px; font-family: sans-serif; transform: scaleX(1.0727);" data-canvas-width="51.65049326599326">Methods</div><div style="left: 93.1212px; top: 718.993px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01751);" data-canvas-width="336.2521239057239">For the research, we used PCR-RT commercial kit Bird-Flu-PCR</div><div style="left: 79.8182px; top: 733.774px; font-size: 12.564px; font-family: serif; transform: scaleX(0.991767);" data-canvas-width="350.62279629629654">(Ukrzoovetprompostach, Ukraine), LAMP (the protocol has been</div><div style="left: 79.8182px; top: 748.555px; font-size: 12.564px; font-family: serif; transform: scaleX(0.992143);" data-canvas-width="255.29490707070704">optimized and patented by SSRILDVSE), QIAamp</div><div style="left: 335.108px; top: 748.887px; font-size: 7.53838px; font-family: serif;">®</div><div style="left: 340.835px; top: 748.555px; font-size: 12.564px; font-family: serif; transform: scaleX(0.9889);" data-canvas-width="87.56712306397306">Viral RNA Mini</div><div style="left: 79.8182px; top: 763.336px; font-size: 12.564px; font-family: serif; transform: scaleX(0.983499);" data-canvas-width="348.58491986531993">Kit. For the study, we used pathological and biological materials from</div><div style="left: 79.8182px; top: 778.117px; font-size: 12.564px; font-family: serif; transform: scaleX(0.973097);" data-canvas-width="348.5145616161615">birds, which were sent to the SSRILDVSE from all regions of Ukraine</div><div style="left: 79.8182px; top: 792.898px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00125);" data-canvas-width="255.80249158249154">according to the 2013–2014 State monitoring plan.</div><div style="left: 93.1212px; top: 807.68px; font-size: 12.564px; font-family: serif; transform: scaleX(1.07037);" data-canvas-width="339.7537031986531">Set up of the real time PCR reactions and parameters of</div><div style="left: 79.8182px; top: 822.461px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00372);" data-canvas-width="278.83225420875414">amplifications are indicated in the instruction to the kit.</div><div style="left: 93.1212px; top: 837.242px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01038);" data-canvas-width="317.68131531986523">The following protocol was used to set up the RT- LAMP: 2.5</div><div style="left: 410.783px; top: 839.628px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 418.022px; top: 837.242px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00008);" data-canvas-width="10.817580808080807">L</div><div style="left: 79.8182px; top: 852.023px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00654);" data-canvas-width="351.46583888888847">10 X Thermopol buffer, 1 mmol/L betaine, 5 mmol/L MgSO4,</div><div style="left: 79.8182px; top: 866.804px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00309);" data-canvas-width="148.0890377104377">1.4 mmol/L - BNTP, 12.5</div><div style="left: 227.872px; top: 869.19px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 235.281px; top: 866.804px; font-size: 12.564px; font-family: serif; transform: scaleX(0.998994);" data-canvas-width="195.88616043771046">mol/L SYBR GREEN, 0.5 mmol/L</div><div style="left: 79.8182px; top: 881.585px; font-size: 12.564px; font-family: serif; transform: scaleX(0.968466);" data-canvas-width="87.53320033670033">MnCL2, up to 25</div><div style="left: 167.376px; top: 883.972px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 174.556px; top: 881.585px; font-size: 12.564px; font-family: serif; transform: scaleX(0.977144);" data-canvas-width="253.66661616161613">L Nuclease-free water, 8 U Bsm DNA polymerase,</div><div style="left: 79.8182px; top: 896.366px; font-size: 12.564px; font-family: serif; transform: scaleX(0.999786);" data-canvas-width="18.845959595959595">0.1</div><div style="left: 98.8736px; top: 898.753px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 106.113px; top: 896.366px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01128);" data-canvas-width="76.94177104377104">M/1 of F3, 0.1</div><div style="left: 183.04px; top: 898.753px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 190.279px; top: 896.366px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01101);" data-canvas-width="78.33637205387205">M/1 of B3, 0.8</div><div style="left: 268.599px; top: 898.753px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 275.838px; top: 896.366px; font-size: 12.564px; font-family: serif; transform: scaleX(1.02822);" data-canvas-width="81.82915656565658">M/1 of FIP, 0.8</div><div style="left: 357.655px; top: 898.753px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 364.894px; top: 896.366px; font-size: 12.564px; font-family: serif; transform: scaleX(1.03296);" data-canvas-width="64.16421043771044">M/1 of BIP,</div><div style="left: 79.8182px; top: 911.148px; font-size: 12.564px; font-family: serif; transform: scaleX(0.999786);" data-canvas-width="18.845959595959595">0.4</div><div style="left: 98.6641px; top: 913.534px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 105.903px; top: 911.148px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00821);" data-canvas-width="122.8505286195286">M/1 of LF, 0.4 of LB, 2</div><div style="left: 228.752px; top: 913.534px; font-size: 12.564px; font-family: sans-serif;">μ</div><div style="left: 235.991px; top: 911.148px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00073);" data-canvas-width="46.750543771043766">L cDNA.</div><div style="left: 93.1212px; top: 925.929px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01628);" data-canvas-width="336.25338030303027">During our work, we used the following optimal temperature and</div><div style="left: 79.8182px; top: 940.71px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00359);" data-canvas-width="250.36229124579123">time for the amplification – 59°C and 60 minutes.</div><div style="left: 93.1212px; top: 955.491px; font-size: 12.564px; font-family: serif; transform: scaleX(0.985987);" data-canvas-width="335.038444107744">The sensitivity of diagnostic kit Bird-Flu-PCR and RT- LAMP was</div><div style="left: 79.8182px; top: 970.272px; font-size: 12.564px; font-family: serif; transform: scaleX(1.03878);" data-canvas-width="349.8438299663301">determined by testing cDNA of the reference strain of AIV H5N1,</div><div style="left: 79.8182px; top: 985.053px; font-size: 12.564px; font-family: serif; transform: scaleX(0.96431);" data-canvas-width="350.4946437710436">which was provided to us by NSC Institute for Experimental and</div><div style="left: 79.8182px; top: 999.834px; font-size: 12.564px; font-family: serif; transform: scaleX(1.03385);" data-canvas-width="349.71819023569003">Clinical Veterinary Medicine (Kharkiv, Ukraine). For the standard,</div><div style="left: 79.8182px; top: 1014.62px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00117);" data-canvas-width="324.16306902356894">we employed concentration in the range of 10.0-0.01 ng/sample.</div><div style="left: 79.8182px; top: 1042.74px; font-size: 12.564px; font-family: sans-serif; transform: scaleX(1.08437);" data-canvas-width="45.38107070707071">Results</div><div style="left: 93.1212px; top: 1056px; font-size: 12.564px; font-family: serif; transform: scaleX(1.07535);" data-canvas-width="43.282887205387205">Table 1.</div><div style="left: 465.606px; top: 296.252px; font-size: 12.564px; font-family: serif; transform: scaleX(1.0077);" data-canvas-width="338.08395117845106">This table shows the reproducibility results obtained by both</div><div style="left: 452.303px; top: 311.033px; font-size: 12.564px; font-family: serif; transform: scaleX(1.01083);" data-canvas-width="349.36137340067336">methods. However, taken into account absence of highly pathogenic</div><div style="left: 452.303px; top: 325.814px; font-size: 12.564px; font-family: serif; transform: scaleX(0.987023);" data-canvas-width="348.5999966329965">avian influenza virus circulating in Ukraine during the studied period,</div><div style="left: 452.303px; top: 340.595px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00281);" data-canvas-width="349.04601767676763">it was not possible to confirm these results with protocols of positive</div><div style="left: 452.303px; top: 355.376px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00166);" data-canvas-width="43.62211447811447">samples.</div><div style="left: 465.606px; top: 370.158px; font-size: 12.564px; font-family: serif; transform: scaleX(1.07535);" data-canvas-width="43.282887205387205">Table 2.</div><div style="left: 465.606px; top: 384.939px; font-size: 12.564px; font-family: serif; transform: scaleX(0.991712);" data-canvas-width="332.5872129629629">It has been established that the sensitivity of PCR-RT kit Bird-Flu-</div><div style="left: 452.303px; top: 399.72px; font-size: 12.564px; font-family: serif; transform: scaleX(0.947204);" data-canvas-width="350.3074405723905">PCR is 0.01 ng/sample for gene M and 0.1 ng/sample for subtype</div><div style="left: 452.303px; top: 414.501px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00091);" data-canvas-width="33.84734343434343">H5N1.</div><div style="left: 465.606px; top: 429.282px; font-size: 12.564px; font-family: serif; transform: scaleX(1.06451);" data-canvas-width="338.3352306397306">Fig. 1. Visual detection of LAMP products with different</div><div style="left: 452.303px; top: 444.063px; font-size: 12.564px; font-family: serif; transform: scaleX(1.04989);" data-canvas-width="345.90502441077433">concentrations of cDNA of avian influenza virus (ng per sample):</div><div style="left: 465.606px; top: 458.844px; font-size: 12.564px; font-family: serif; transform: scaleX(0.98714);" data-canvas-width="332.51811111111107">1 – 10; 2 – 5; 3 – 1.0; 4 – 0.1; 5–7 – 0.01; 8–9 – 0.1; 10 – negative.</div><div style="left: 465.606px; top: 473.626px; font-size: 12.564px; font-family: serif; transform: scaleX(0.973046);" data-canvas-width="336.96575757575755">We have examined the LAMP results using electrophoresis for</div><div style="left: 452.303px; top: 488.407px; font-size: 12.564px; font-family: serif; transform: scaleX(1.0168);" data-canvas-width="349.49078232323234">the confirmation of visual detection and correct interpretation of the</div><div style="left: 452.303px; top: 503.188px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00081);" data-canvas-width="76.7784393939394">results (Fig. 2).</div><div style="left: 465.606px; top: 517.969px; font-size: 12.564px; font-family: serif; transform: scaleX(1.11794);" data-canvas-width="339.61047390572384">Fig.2. Electrophoresis results for LAMP products. M –</div><div style="left: 452.303px; top: 532.75px; font-size: 12.564px; font-family: serif; transform: scaleX(1.07287);" data-canvas-width="349.6302424242423">molecular weight marker; 1 – 10.0; 2 – 5.0; 3 – 1.0; 4 – 0.1; 5–7</div><div style="left: 452.303px; top: 547.531px; font-size: 12.564px; font-family: serif; transform: scaleX(1.04157);" data-canvas-width="141.6713602693603">– 0.01; 8 - negative control</div><div style="left: 593.967px; top: 547.531px; font-size: 12.564px; font-family: serif;">.</div><div style="left: 465.606px; top: 562.313px; font-size: 12.564px; font-family: serif; transform: scaleX(1.11226);" data-canvas-width="340.6495144781143">It has been established that the sensitivity of LAMP is</div><div style="left: 452.303px; top: 577.094px; font-size: 12.564px; font-family: serif; transform: scaleX(0.971465);" data-canvas-width="350.35895286195273">0.1 ng/sample. Slightly lower sensitivity of LAMP in comparison</div><div style="left: 452.303px; top: 591.875px; font-size: 12.564px; font-family: serif; transform: scaleX(1.03848);" data-canvas-width="349.743318181818">to PCR-RT can be explained by visual detection of the products of</div><div style="left: 452.303px; top: 606.656px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00114);" data-canvas-width="100.14742929292929">the LAMP reaction.</div><div style="left: 452.303px; top: 634.783px; font-size: 12.564px; font-family: sans-serif; transform: scaleX(1.10256);" data-canvas-width="75.39640235690236">Conclusions</div><div style="left: 465.606px; top: 648.043px; font-size: 12.564px; font-family: serif; transform: scaleX(1.00082);" data-canvas-width="191.27392592592594">1. Sensitivity of both methods is high.</div><div style="left: 465.606px; top: 662.824px; font-size: 12.564px; font-family: serif; transform: scaleX(1.04028);" data-canvas-width="336.56371043771037">2. LAMP is a perspective screening method for the diagnosis of</div><div style="left: 452.303px; top: 677.605px; font-size: 12.564px; font-family: serif; transform: scaleX(0.993138);" data-canvas-width="348.65779090909086">viral infectious diseases supported by confirmation of positive results</div><div style="left: 452.303px; top: 692.387px; font-size: 12.564px; font-family: serif; transform: scaleX(1.02835);" data-canvas-width="62.832429292929284">by PCR-RT.</div> ER -