Name: evaluation of a universal nested reverse transcription polymerase chain reaction for the detection of lyssaviruses
Uploaded: 2019-05-02T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses at JoVE.com

The study was supported by the National Key Research and Development Plan (Grant no. 2016YFD0500401) and the National Natural Science Foundation of China (Grant no. 31302043).

The use of mice in this protocol was approved by the Administrative Committee on Animal Welfare of the Institute of Military Veterinary Medicine, the Academy of Military Medical Sciences, China (Laboratory Animal Care and Use Committee Authorization, permit number JSY-DW-2010-02). All institutional and national guidelines for the care and use of laboratory animals were followed.
1. RNA Extraction
<ol>
	<li>Extract RNA from rabies-suspected brain tissue, skin biopsies, saliva, or CSF or from ...

<ol>
	<li>Hemachudha, T., Laothamatas, J., Rupprecht, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+rabies:+a+disease+of+complex+neuropathogenetic+mechanisms+and+diagnostic+challenges.">Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges.</a> Lancet Neurology. 1 (2), 101-109 (2002).</li><li>Amarasinghe, G. K., Arechiga Ceballos, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxonomy+of+the+order+Mononegavirales:+update+2018.">Taxonomy of the order Mononegavirales: update 2018.</a> Archives of Virology. 163 (8), 2283-2294 (2018).</li><li>Nokireki, T., Tammiranta, N., Kokkonen, U. M., Kantala, T., Gadd, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tentative+novel+lyssavirus+in+a+bat+in+Finland.">Tentative novel lyssavirus in a bat in Finland.</a> Transboundary and Emerging Diseases. 65 (3), 593-596 (2018).</li><li>Hu, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyssavirus+in+Japanese+Pipistrelle,+Taiwan.">Lyssavirus in Japanese Pipistrelle, Taiwan.</a> Emerging Infectious Diseases. 24 (4), 782-785 (2018).</li><li>Rupprecht, C. E., Fooks, A. R., Abela-Ridder, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+techniques+in+rabies.">Laboratory techniques in rabies.</a> World Health Organization. , 74-195 (2018).</li><li>Mani, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+real-time+Taqman+PCR+for+antemortem+and+postmortem+diagnosis+of+human+rabies.">Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies.</a> Journal of Medical Virology. 86 (10), 1804-1812 (2014).</li><li>Fooks, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+for+the+detection+of+rabies+virus:+challenges+and+hopes+in+the+21st+century.">Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century.</a> PLOS Neglected Tropical Diseases. 3 (9), 530 (2009).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+outbreak+of+pig+rabies+in+Hunan+province,+China.">An outbreak of pig rabies in Hunan province, China.</a> Epidemiology and Infection. 136 (4), 504-508 (2008).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+complete+genome+of+the+first+Irkut+virus+isolate+from+China:+comparison+across+the+Lyssavirus+genus.">Analysis of the complete genome of the first Irkut virus isolate from China: comparison across the Lyssavirus genus.</a> Molecular Phylogenetics and Evolution. 69 (3), 687-693 (2013).</li><li>Chen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+Transmission+of+Irkut+Virus+from+Dogs+to+Humans.">Possible Transmission of Irkut Virus from Dogs to Humans.</a> Biomedical and Environmental Sciences. 31 (2), 146-148 (2018).</li><li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+outbreaks+caused+by+steppe-type+rabies+viruses+in+China.">Disease outbreaks caused by steppe-type rabies viruses in China.</a> Epidemiology and Infection. 143 (6), 1287-1291 (2015).</li><li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Livestock+rabies+outbreaks+in+Shanxi+province,+China.">Livestock rabies outbreaks in Shanxi province, China.</a> Archives of Virology. 161 (10), 2851-2854 (2016).</li></ol>

Currently, RABV is a major lyssavirus responsible for nearly all human and animal rabies in China, as well as in other countries. In addition, an IRKV variant was first identified from a Murina leucogaster bat in the Jilin province in Northeast China in 201210, and it has been reported to cause a dog&#8217;s death in the Liaoning Province in 201711. Most recently, a novel lyssavirus, TWBLV, was also identified from a Japanese pipistrelle bat in Taiwan, Chi...

Rabies is a worldwide zoonotic disease caused by viruses within the genus Lyssavirus1. Lyssaviruses (family&#160;Rhabdoviridae) are single-negative-stranded RNA viruses with an approximately 12 kb genome that encodes five proteins: N, phosphoprotein (P), matrix protein (M), glycoprotein (G), and the large protein or polymerase (L).&#160;Based on nucleotide sequences of the N gene, genetic distance, and antigenic patterns, the lyssaviruses have been divided into 16 species, comprising classical rabies virus (RABV) and the rabies-related viruses (RRV): Lagos bat virus (LBV), Duvenhage virus (DUVV), Mokola virus (MOKV), European bat lyssavirus 1 (EBLV-1), European bat lyssavirus 2 (EBLV-2), Australian bat lyssavirus (ABLV), Aravan virus (ARAV), Ikoma virus (IKOV), Bokeloh bat lyssavirus (BBLV), Gannoruwa bat lyssavirus (GBLV), Irkut virus (IRKV), Khujand virus (KHUV), West Caucasian bat virus (WCBV), Shimoni bat virus (SHIBV), and Lleida bat lyssavirus (LLEBV)2. Recently, two additional lyssaviruses have been identified: Kotalahti bat lyssavirus (KBLV) isolated from a Brandt&#8217;s bat (Myotis brandtii) in Finland in 20173 and Taiwan bat lyssavirus (TWBLV) isolated from a Japanese pipistrelle (Pipistrellus abramus) in Taiwan, China in 2016&#8211;20174.
All mammals are susceptible to rabies; however, no gross pathognomonic lesions or specific clinical signs permit its identification, and diagnosis can only be made in the laboratory5. The most widely used method for rabies diagnosis is the FAT, which is considered as the gold standard by both the WHO and the OIE5,6. Nevertheless, the FAT can produce unreliable results on degraded/autolyzed brain tissue samples. Additionally, it cannot be used to assay biological fluid specimens such as cerebrospinal fluid (CSF), saliva, and urine, thereby largely precluding its employment in antemortem diagnosis7. Alternative conventional diagnostic tests, such as the rabies tissue culture infection test (RTCIT) and the mouse inoculation test (MIT), require several days6, a major drawback when a rapid diagnosis is essential.
Various molecular diagnostic tests (e.g., the detection of viral RNA by RT-PCR, the PCR&#8211;enzyme-linked immunosorbent assay [PCR-ELISA], in situ hybridization, and real-time PCR) are used as rapid and sensitive techniques for rabies diagnosis8. RT-PCR is now recommended by OIE for routine rabies diagnosis, and a heminested (hn) PCR is described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals to detect all lyssaviruses5. Here we describe a pan-lyssavirus nested RT-PCR, which allows the specific and sensitive detection of all 18 lyssavirus species comparable to or exceeding that obtained by the FAT. The principle of the method is an RT of the target RNA (conserved region of the lyssavirus N gene) into cDNA, followed by the amplification of the cDNA by two rounds of PCR. The cDNA undergoes the first-round PCR with outer primers to amplify an 845 bp fragment; then, the second-round PCR uses the first-round PCR product as a template to amplify a 371 bp fragment with inner primers. The two rounds of PCR significantly increase the sensitivity of the assay.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">50 &#215; TAE</td>
			<td style="width:177px;">Various</td>
			<td style="width:108px;">Various</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">6 &#215; loading buffer</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">9156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Agarose</td>
			<td style="width:177px;">US Everbright&#174; Inc</td>
			<td style="width:108px;">A-2015-100g</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:260px;">ddH2O</td>
			<td style="width:177px;">Various</td>
			<td style="width:108px;">Various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">DL 2,000 Marker</td>
			<td style="width:177px;">Takara</td>
			<td style="width:108px;">3427A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">dNTPs (10 mM)</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">4019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">dNTPs (2.5 mM)</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">4030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Electrophoresis System</td>
			<td style="width:177px;">Tanon</td>
			<td style="width:108px;">EPS300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Ex-Taq (5 U/&#956;L)</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">RR001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Gel Imaging System</td>
			<td style="width:177px;">UVITEC</td>
			<td style="width:108px;">Fire Reader</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Microcentifuge tubes</td>
			<td style="width:177px;">Various</td>
			<td style="width:108px;">Various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">M-MLV reverse transcriptase (200 IU/&#181;L)</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">2641A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">NanoDrop 1000 Spectrophotometer</td>
			<td style="width:177px;">Thermoscientific</td>
			<td style="width:108px;">ND1000</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:260px;">Oligo (dT)15</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">3805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">PCR Machine</td>
			<td style="width:177px;">BIO-RAD</td>
			<td style="width:108px;">T100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">PCR Tubes</td>
			<td style="width:177px;">Various</td>
			<td style="width:108px;">Various</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">Phusion High-Fidelity DNA Polymearase</td>
			<td style="width:177px;">NEW ENGLAND BioLabs</td>
			<td style="width:108px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Pipettors</td>
			<td style="width:177px;">Various</td>
			<td style="width:108px;">Various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Random Primer</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">3801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase Inhibitor (40 IU/&#181;L)</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">2313A</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:260px;">RNase-free ddH2O</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">9102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Taq Buffer (10&#215;)</td>
			<td style="width:177px;">TakaRa</td>
			<td style="width:108px;">9152A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Tips</td>
			<td style="width:177px;">Various</td>
			<td style="width:108px;">Various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Vortex mixer</td>
			<td style="width:177px;">Various</td>
			<td style="width:108px;">Various</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of a universal nested reverse transcription polymerase chain reaction for the detection of lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

Results of nested RT-PCR to detect 18 lyssavirus species are shown in Figure 1. All PCR positive controls showed the expected 845 bp in the first- and 371 bp in the second-round amplifications with no band in the negative control. All 18 lyssaviruses produced the expected 845 and/or 371 bp bands, indicating that the nested RT-PCR detected all 18 lyssaviruses. Sixteen lyssaviruses plasmids had efficient amplification in two rounds of PCR, but two, namely ARAV and IKOV, had amplification in ei...

Watch this Scientific Journal Video about Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses at JoVE.com

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

The nested RT-PCR has been developed to detect all Lyssaviruses, including all 16 ICTV approved and two novel Lyssavirus species. The validation of this method has been proved by using over 9000 animal brain specimens in 10 years of clinical rabies diagnosis and surveillance in China. The nested RT-PCR not only can produce credible result on fresh brain tissue samples, but also on degraded samples.The inundation this method can be used to assay biological fluid specimens such as cerebrospinal fluid, saliva, and urine, thereby this method can be applied into ultimatum diagnoses. In order to minimize PCR carry-over contamination, it is important to prepare negative and positive controls It's really important to take steps. For example, for each RNA extraction needs preparation, PCR set up, and gel electrophoresis should be operated in physically separate areas.It is also recommended to use spiral tapes in RNA extraction and reverse transcription in order to prevent rabies contamination to the peptide chain. Proper handling such as preparing your agents on ice, changing gloves regularly, and use of ribonuclease free materials will prevent the introduction of ribonuclease and eliminate degradation of RNA. To begin this procedure, extract the RNA from rabies suspected tissue, skin biopsies, saliva, cerebrospinal fluid or RABV infected cell culture as outlined in the text protocol.When ready to proceed, removed the needed reagents from the freezer and keep them on ice. Make sure to thaw and vortex each reagent before use. For reverse transcription of the viral RNA to cDNA prepare 12 micro liters of reaction mix for each sample on ice and a 1.5 milliliter centrifuge tube as outlined in Table 1 of the text protocol in a cleanroom.To account for pipetting variations, prepare a volume of master mix at least one reaction size greater than required, and then divide the reaction mix into 0.2 milliliter PCR tubes. In a PCR workstation in a template room, add eight microliters of either the sample or one of the controls to the reaction mix. The positive control is RNA extracted from the cell culture infected with fixed-RABV strain CVS11, while the negative control contains Rnase-free double distilled water.Mix the contents of each tube by first vortexing and then by briefly centrifuging. After this, load the reaction tubes into a thermocycler. Set up and run the cDNA synthesis program as outlined in the text protocol.First, retrieve the necessary reagents and keep them on ice in a cleanroom until ready to use. When ready, thaw and vortex the reagents. Next, prepare 48 microliters of first round PCR mix for each sample in a 1.5 milliliter centrifuge tube as outlined in Table 2 of the text protocol and divide the reaction mix into 0.2 milliliter PCR tubes.In a PCR workstation in a template room, add a two microliter sample of the cDNA into the first round PCR mix. Include two microliters of CVS11 cDNA as a positive control, and two microliters of double distilled water as a negative control. Then, transfer the sealed tubes into a PCR thermocycler and cycle using the parameters listed in Table 3 of the text protocol.To begin, prepare 48 microliters of second round PCR mix for each sample in a 1.5 milliliter centrifuge tube as outlined in Table 4 of the text protocol and divide the reaction mix into 0.2 milliliter PCR tubes. Add two microliters of the first round PCR product into the second round PCR mix. Include double distilled water as a negative control for this round of PCR.Then, perform PCR thermocycling using the parameters listed in Table 3 of the text protocol. First, add 1.5 grams of agarose to 100 milliliters of Tris-acetate EDTA and heat it in a microwave oven to dissolve it thoroughly. Next, add ethidium bromide at a final concentration of 0.01%Pour the gel into the mold and leave it to solidify at ambient temperature for at least 30 minutes.Prepare the loading samples by mixing five microliters of each PCR product with one microliter of 6x loading buffer. Separately load the samples and suitable DNA marker into the wells and run the gel for approximately 30 to 45 minutes at 120 volts until the dye line is approximately 75 to 80%down the gel. When the run is complete, turn off the power and disconnect the electrodes from the power source.Then, carefully remove the gel from the gel box. Use a UV gel documenting device to visualize and photograph the DNA fragments. Characterize the nested reverse-transcription PCR as outlined in the text protocol.The results of a representative nested RT-PCR to detect 18 Lyssavirus species is shown here. The PCR positive controls show the expected band at 845 base pairs in the first round amplification and at 371 base pairs for the second. No band is seen for the negative controls.All 18 Lyssaviruses are seen to produce the same two bands as the positive control, indicating that the nested RT-PCR detected all 18 Lyssaviruses. While 16 of the plasmids have efficient amplification in two rounds of PCR, two of them, Aravan virus and Ikoma virus, are amplified only in either the first or second round, respectively. The sensitivity of the method varies in the detection of different Lyssavirus plasmids, from values ranging from 2.24 to 2, 240 molecules per microliter.These differences can be attributed to the mismatches between the primers and templates due to viral sequence diversity. A comparison of 9, 624 brain tissues from clinical specimens that are tested by nested PCR versus those tested with FAT shows that nested RT-PCR as a sensitivity of 100%and a specificity of 99.97%The accordance between the two methods is 99.07%Ten other rabies laboratories were invited to conduct the test to further validate the nested RT-PCR. All ten laboratories obtained nested RT-PCR results that are 100%in accordance with the FAT, with no false negatives or false positives, indicating that the nested RT-PCR has a high specificity and reproducibility.RT-PCR is now recommended by OIE for using rabies technologies. And our nested RT-PCR has been proved as the national standard of rabies technologies in China in 2018.

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