Use of reverse transcriptase loop-mediated isothermal amplification assay for field detection of Newcastle disease virus using less invasive samples

A novel nucleic acid amplification method, loop-mediated isothermal amplification, was developed and recently demonstrated detection of Newcastle disease virus (NDV) in tissue samples. But slaughter of poultry for test samples is often faced with resentment by low-income farmers. This study was undertaken to determine the test properties of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) in detection of NDV in clinical cases using cloacal and oropharyngeal swabs. Samples included 46 tracheal tissues, 94 cloacal and 107 oro-pharyngeal swabs from on-station and 30 spleens, 74 cloacal and 74 oro-pharyngeal swabs from the field. Analysis was done using specific RT-LAMP targeting the fusion (F) protein. While the method detected NDV from swab samples, no RNA of other poultry disease viruses was amplified, indicating analytical specificity of 100%. RT-LAMP took ≤36 minutes in 83% (n=329) of positive reactions with all samples amplified in <60 minutes. Results were easily observed with a naked eye. Cloacal and oro-pharyngeal swabs could be a convenient and cheaper alternative in diagnosis of NDV infection by RT-LAMP in resource poor countries.


Introduction
PCR) is also gradually being introduced.HI test is labour-intensive requiring serial dilution of serum Newcastle disease (ND) is the principal factor samples, standardization of viruses and manual limiting rural poultry production in Asian and African reading of plates (Rothbarth et al., 1999).The test countriesincluding Uganda.The disease may kill up to needs two samples, with atleast two weeks interval to 80% of household poultry and is caused by enveloped differentiate between antibodies due to vaccination or RNA viruses of the Avian Paramyxovirus serotype 1 active infection (Blomstrom et al., 2008).Similarly, (APMV-1) (Alexander 2003).To prevent spread of the requirements for running RT-PCR including Newcastle disease virus (NDV) during outbreaks, reaction time of at least 90 minutes make RT-PCR faster methods of diagnosis are required (IAEA /FAO, labourious, time consuming and expensive (Rychlik et 2006).Virus isolation is the gold standard method for al .,1990;Pavlov et al., 2004).About a decade ago, a detection of NDV, although haemagglutination inhibition novel nucleic acid amplification method called loop-(HI) test is considered the standard laboratory test for mediated isothermal amplification (LAMP) was developed (Notomi et al., 2000).The method can the diagnosis of ND (OIE, 2010).amplify a tiny amount of either Deoxyribonucleic acid In Uganda, diagnosis of ND is often based on (DNA) or Ribonucleic Acid (RNA) rapidly and clinical history, clinical signs, lesions and laboratory specifically under isothermal conditions with high tests mainly Haemagglutination (HA) and immunosensitivity and specificity (Nagamine et al., 2002a).chromatographic assay (ICA) strips.Diagnosis by This method is performed using the Bst DNA reverse transcriptase polymerase chain reaction (RT-polymerase large fragment and requires four or six cloacal and 107 oro-pharyngeal swabs were taken primers targeting six or eight distinct sequences, from experimental chicks while field samples respectively, on the target gene.Recently, it was included 30 spleen tissues, 74 cloacal and 74 orodemonstrated that the LAMP-based assay can detect pharyngeal swabs.Clinical signs and lesions as NDV in tissue samples rapidly and with high described in Calnek et al. (1997) and ICA were used to sensitivity and specificity (Pham et al., 2005).Since confirm NDV infection before sample collection.ICA ® development of more convenient and rapid methods (Anigen Rapid NDV Ag Test Kit , Animal Genetics, for the diagnosis of diseases is being encouraged Inc., Korea) was used according to manufacturer's (IAEA/FAO, 2006), this study was conducted to instructions (www.anigen.co.kr.com).Positive test assess the potential of LAMP in detection of NDV control samples included five each of the bursa of from cloacal and oro-pharyngeal swabs in clinical fabricius and lungs for infectious bursal disease virus cases of experimental and natural infection.
(IBDV) and infectious bronchitis virus (IBV), respectively, and spleens from the control group for cryogenic vials (Ambion, Applied Biosystems, Experimental chickens: A total of 230 male layer Woodward, USA).The eluted RNA, previously stored o day-old chicks obtained from a commercial breeding at -80 C, was later precipitated using the ethanol farm, Bokomo Breeders Company (Uganda) were precipitation of nucleic acids procedures (http://open used in an on-station experiment.Chicks were introduced wetware.org/wiki/Ethanol_precipitation_of_nucleic_ at day 1 of age, kept under biosecure conditions and acids).The method involved the use of absolute fed on commercial feeds.No vaccination was done, ethanol (99.7% ethanol), sodium acetate and treatment only involved use of coccidiostats and a refrigerated centrifuge.Precipitation was undertaken decline in maternal antibodies against ND was in order to facilitated shipment of RNA samples at a monitored for up to five weeks.Blood was collected o o temperature of about 20 C to 22 C to Obihiro from a cumulative total of 82 randomly selected chicks University of Agriculture and Veterinary Medicine, at a proportion of 10% of the flock weekly for five Japan, where RT-LAMP analysis was later done after weeks.Serum was tested for antibodies titres against re-dissolving in sterile RNAase-free double-distilled ND by HI test (OIE, 2010).
water, vortexed and span down to re-suspend.

Virus isolates: Sixteen velogenic NDV isolates from
RT-LAMP analysis: The specific oligonucleotide Eastern Uganda (Otim et al., 2004) were used for primers used were designed for the NDV conserved experimental infection.Virus passaging and HA test region using NDV strain ASTR/74 (Accession were done as described in the Manual of Diagnostic number: Y19016) to target the complete genome of the Tests and Vaccines for Terrestrial Animals 2010 (OIE, fusion (F) protein.The fusion (F antigen) protein is the 2010).
most important glycoprotein in pathogenicity and virulence of the NDV as it provides the penetration Virus inoculation: At five weeks of age, 138 chicks function for the virus particle and is also required for were inoculated with 0.3 ml of a 10% diluted NDVfusion between infected and adjacent non-infected laden allantoic fluid by intranasal and ocular routes as cells (Nagai et al., 1989).Primer design was done earlier on described (Steel et al., 2009).Chicks were using primer explorer V4 software (http:// then monitored for clinical signs.Ten chicks (not primerexplorer.jp/elamp4.0.0/index.html).Sequences inoculated) formed the control group.
of the primer set (named UGA2 in this study) are: Sample collection: Tracheal tissues, cloacal and oro-FIP-5'CGGGTCCTCACCCAAAGATGTTGGTG pharyngeal swabs were collected at peak of clinical GAAAGCGCGTACAG-3'; signs.Collection of blood and postmortem examination BIP-5'ACACTCATGGGGGCCGAAGGTGAAGA were conducted as described by Hofstad et al. (1984) CCCTCGTTGGTACA3'; and Fowler (1996).Forty six tracheal tissues, 94  The primer set specifically amplified the NDV RNA but did not amplify RNA of infectious bursal Determination of relative diagnostic sensitivity disease virus (IBDV), infectious bronchitis virus and specificity: Relative diagnostic sensitivity (Se) (IBV) or that of host chicken.The primers neither and specificity (Sp) of RT-LAMP of cloacal and oroamplified RNA free water (Fig. 1).pharyngeal swabs were determined by comparing the test using each of the sample categories against ICA RT-LAMP detection of Newcastle disease virus in (Martin et al., 1987).A kappa statistic was calculated experimental samples: Of the 46 RNA samples to determine the level of agreement (Martin et al., from tracheal tissues, 45 (97.8%) were positive just as 1987) and in each case, this was done after determining 93/94 (98.9%) and 106/107 (99.1%) from cloacal and whether or not a test bias existed.Existence of a test oro-pharyngeal swabs, respectively.A deliberate bias was assessed by a non-parametric statistic, comparison of results of RT-LAMP made for 37 sets of McNemar's test (Motulsky, 1995).
samples, each of cloacal and oro-pharyngeal collected from randomly selected chicks among those with

Results
prominent ND signs produced similar results.It was Clinical signs among infected chicks: On day 3, observed that RNA amplification achieved in post-inoculation chicks started showing signs of NDV agreement in100% of sets of swabs.infection as described by Calnek et al. (1997) with RT-LAMP detection of Newcastle disease virus in morbidity and mortality rates of 100% and 93.5%, field samples: There was variation in results when 74 respectively, had been recorded by the end of the fifth cloacal and 74 oro-pharyngeal clinical samples were day of clinical disease.Only nine out of the 138 analysed using ICA, a real-time turbidimeter and by a absence of agreement between the two tests.heat block.In Table 1, results of ICA, RT-LAMP by Discussion turbidimeter and a heat block are shown.Whereas positive results of cloacal swabs by both ICA and RT-ND is an economically important disease affecting LAMP (with a naked eye) were similar (97.3%), these chickens, particularly in developing countries including were however lower than 100.0%positive results Uganda (Mukiibi, 1992), is still endemic in the observed in RT-LAMP (using a turbidimeter).country (Otim et al., 2007) and chickens in many Conversely, results of oro-pharyngeal samples by RT-villages remain fully susceptible to infection with LAMP deduced by a naked eye were lower (97.3%)virulent NDV (Mukiibi-Muka and Olaho-Mukani, than those of RT-LAMP turbidimetry (98.6%), though 1998).Effective vaccines against NDV infections have higher than those of ICA (94.6%).Results of samples been developed and are widely applied for prevention from non-ND samples (IBDV and IBV) and those of the disease worldwide in both commercial and free from healthy host chickens were similar (0.0%) for all ranging production systems (Thekisoe et al., 2004).three tests.RNA of all 30 (100%) spleen samples from However, accurate screening of samples before field clinical cases was positive by RT-LAMP on vaccination and during outbreaks is of paramount either heat block or turbidimeter (results not shown).importance.Whereas the use of clinical signs, haema-In another comparison, RT-LAMP detected NDV in all glutination inhibition and chromatographic tests are (100%, n=74) cloacal samples taken from all ND widely applied to confirm the presence or absence of clinical cases regardless of age group while the NDV infections, development of more convenient and detection proportion of ICA was only 96% (results not rapid methods for the diagnosis of diseases is being shown), specifically lower in chicks than adult chickens.encouraged (IAEA/FAO,2006).
The RT-LAMP assay targeting the F protein of

Relative sensitivity and specificity of RT-LAMP
the Ugandan strain of NDV specifically amplified the using less invasive samples: A comparison of RNA of the virus and amplified neither RNA of other NDV detection by ICA with either of RT-LAMP for avian viral infections commonly occurring chickens in cloacal swabs or oropharyngeal swabs had relative the country including IBDV and IBV nor RNA from sensitivity/specificity of 100%/33% for cloacal swabs un-infected host chicken.A field sample detection rate and 100%/67% for oro-pharyngeal swabs, respectively of >99% and >97% of RT-LAMP by turbidimetry and (Table -2).Calculating the level of agreement (κ), was with a naked eye, respectively, were fair for swab of limited value due existence of bias in the comparison samples.Unlike the study by Pham et al.(2005) in of ICA with RT-LAMP using cloacal swabs.Due to which no NDV detection was made from spleen absence of test bias, κ of 0.1 was computed for RTsamples, in this study RT-LAMP amplified RNA from LAMP using oro-pharyngeal swabs, which indicted Our study has demonstrated that RT-LAMP can samples have also demonstrated the ease of distinguidetect NDV from cloacal and oro-pharyngeal swabs shing between positive and negative reactions based and in less than 60 minutes with samples amplified on turbidity or colour change using the naked eye using a heat block.The method amplifies RNA from immediately after the reaction.Ease of differentiating these specimens with high relative sensitivity (98positive from negative samples after a LAMP reaction 100%) and analytical specificity (100%), yet results with a naked eye or under ultra violet light has been can easily be observed with a naked eye.Use of less extensively reported in the recent past (Tomita et al., invasive samples, the short reaction time, the simple 2008).RT-LAMP assay amplified samples obtained amplification equipment involved, ease of interpretation both in experimental infection and natural exposure of results and the high level of analytical and relative within 60 minutes at a constant isothermal reaction sensitivity and specificity make RT-LAMP a good, o temperature of 64 C using simple heating devices.
convenient and inexpensive alternative in diagnosis of The LAMP assay is, therefore, rapid and simpler NDV in chickens in resource poor countries.Collection to use than RT-PCR, since the later requires thermal of cloacal and oro-pharyngeal swabs does not cause profiling and at least 90 minutes to run a reaction farmer resentment, which makes sample collection (Rychlik et al., 1990;Pavlov et al., 2004).Cloacal and easier in different ND outbreaks.oro-pharyngeal swabs, which are less invasive

F3- 5 '
CAAGCCTGGGCGGTTC3'; infected chicks survived.ND lesions(Calnek et al.,  1997)  were observed in 90.5% (n=63) of internal B3-5'ACAATAAGGCGGGGGAGAA3'; organs in cases in which postmortem was done.All LF-5'ACACTTTGATGGATAAGATAAC3' and chicks in the control group did not show signs or LB-5'CAGAGTTCTCACAGTAGGGA3'.lesions of ND during the experimental period and none Primers were first optimised at different reaction had died by time of termination of study.0 0 temperatures ranging from 60 C-65 C for 90 minutes.Analytical specificity and sensitivity of RT-Specificity tests were conducted against poultry RNA LAMP: Using a primer set (UGA 2) at an optimal viruses known to infect chickens in Uganda including 0 reaction temperature of 64 C, NDV RNA was IBDV and IBV, and host chicken RNA.Loopamp® amplified in 20 to 36 minutes in three repetitions.RNA amplification kit (Eiken Chemical Co, Tokyo, Japan) was used in all RT-LAMP reactions and incubation done on a heat block or in a turbidimeter.In cases where visualization was to be done under ultra violet light, a fluorescent detection reagent (FD) (Eiken Chemical Co., Ltd.) was added into the reaction tubes.The heat block was a thermocycler (GeneAmp, PCR System 9700, Applied Biosystem, 0 Singapore), which was set at 64 C for 60 minutes 0 (amplification time) and 80 C for three minutes (to terminate the elongation and entire reaction) and held 0 at 4 C for short-term storage.Conversely, a real-time LAMP turbidimeter (Loopamp LA 200, Teramics, Japan) was the other incubation equipment used.Reaction products from both methods were read immediately after termination of each reaction.

Fig. 1 .
Fig. 1.Specificity of RT-LAMP with Newcastle Disease VirusResults from the heat block were visualised by the

Table - 2. Relative sensitivity and specificity of RT-LAMP using cloacal and oro-pharyngeal swabs
(Martin et al., 1987)nscriptase loop-mediated spleen tissues in all (100 %, n=30) reactions.This poor degree of agreement between the two tests, with result was not surprising since internal organs including RT-LAMP being superior.Like reported in previous spleen have been reported of a good viral load during similar studies(Pham et al., 2005; Xue et al., 2009; Li infection with velogenic NDV, especially up to 7-10 et al., 2009), RT-LAMP using clocal and orodays post infection (Kommers et al., 2002; Okwor, pharyngeal swabs did not amplify RNA from other 2007).Detecting NDV in spleen could most likely be viral infections and hence analytical specificity of attributed to strain difference in tissue tropism of the 100%.The level of relative diagnostic sensitivity and velogenic (used in our study) compared with the specificity demonstrated by RT-LAMP using cloacal lentogenic strain that was used byPham et al. (2005).andoro-pharyngealswabs is a good method for The good detection rates achieved with cloacal and epidemio-logical studies.Generally, at diagnostic oro-pharyngeal swabs demonstrated good RNA recovery sensitivity and specificity of 99% and 99%, by the two specimen categories.Swabs produced good respectively, a test is regarded relatively good for RNA recovery rate because they were taken at the epidemiological studies(Martin et al., 1987).most appropriate time (about 2-5 days) post-infection,