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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2009 May 13;47(7):2114–2123. doi: 10.1128/JCM.01652-08

Real-Time PCR-Based Pathotyping of Newcastle Disease Virus by Use of TaqMan Minor Groove Binder Probes▿

T Farkas 1,2,*, É Székely 1, S Belák 3, I Kiss 1,3
PMCID: PMC2708511  PMID: 19439542

Abstract

A real-time reverse-transcription PCR was developed to detect and pathotype Newcastle disease viruses (NDV) in clinical samples. Degenerate oligonucleotide primers and TaqMan probes with nonfluorescent minor groove binder (MGB) quencher amplified and hybridized to a region in the fusion protein (F) gene that corresponds to the cleavage site of the F0 precursor, which is a key determinant of NDV pathogenicity. The application of degenerate primers and TaqMan MGB probes provided high specificity to the assay, as was shown by the successful and rapid pathotype determination of 39 NDV strains representing all the known genotypes (I to VIII) and pathotypes (lentogens/mesogens/velogens). The PCR assays specific for lentogenic and velogenic/mesogenic strains had high analytical sensitivity, detecting approximately 10 and 20 copies of the target molecule per reaction, respectively. The detection limit was also determined in terms of 50% egg infective dose (EID50) by using dilution series of virus stock solutions to be approximately 101.0 and 10−1.3 EID50/ml for lentogens and velogens/mesogens, respectively. Organ, swab, and stool specimens from experimentally infected animals were tested to prove the clinical suitability of the method. The results of this study suggest that the described real-time PCR assay has the potential to be used for the rapid detection/pathotyping of NDV isolates and qualitative/quantitative measurement of the virus load.

Newcastle disease (ND) is an infectious viral disease of birds that has a worldwide distribution and a serious economic impact on poultry production. The etiologic agent of the disease is a member of the avian paramyxoviruses (APMV), which are classified into the Avulavirus genus and Paramyxoviridae family of the order Mononegavirales (1, 36). Paramyxoviruses isolated from avian species have been grouped into nine serotypes (APMV-1 to APMV-9), and ND virus (NDV) is referred to as APMV-1 (5). NDV is an enveloped virus that contains a nonsegmented, single-stranded, negative-sense RNA genome of ca. 15 kb coding for six major proteins: a large RNA polymerase (L), hemagglutinin-neuraminidase protein (HN), fusion protein (F), matrix protein (M), phosphoprotein (P), and nucleoprotein (NP), in the order 3′-NP-P-M-F-HN-L-5′ (3, 19, 38). Two additional proteins, V and W, are expressed by mRNAs derived from the P gene via RNA editing (26). The three proteins are amino coterminal and vary in the length and amino acid composition of their carboxy-terminals ends. The V protein is known to have an inhibitory effect on the alpha/beta interferon response of the avian host.

NDV has a wide host range; more than 250 avian species in 27 orders have been reported to be susceptible to the disease (3, 28). The severity of clinical signs depends on the virulence properties of the causative virus and the species, age, immune status, concurrent diseases, and susceptibility of the avian host. Of domestic poultry, chickens are the most susceptible and waterfowl are the least susceptible to ND (5).

The virulence of NDV isolates was traditionally determined on the basis of chicken embryo mortality after allantoic inoculation, and strains were categorized into three main pathotypes (lentogenic, mesogenic, and velogenic); afterward, further categories were created according to the clinical signs observed in infected chickens (6): (i) viscerotropic velogenic, a highly virulent form causing hemorrhagic intestinal lesions; (ii) neurotropic velogenic, a highly virulent form accompanied by a high mortality rate and causing respiratory/nervous signs; (iii) mesogenic, a form with intermediate virulence that also results in respiratory/nervous signs but with a significantly lower mortality rate; (iv) lentogenic, a form with low virulence causing mild or subclinical infection in the respiratory tract; and (v) asymptomatic enteric, an avirulent form leading to an inapparent intestinal infection.

The high variability in virulence and clinical signs, however, made it necessary to define ND unambiguously, e.g., for the purposes of trade and control measures and policies. According to the current World Organization for Animal Health (Office International des Epizooties [OIE]) definition, the assessment of virus virulence is based on the intracerebral pathogenicity index (ICPI) test and the amino acid sequence at the F0 protein cleavage site. Infections of poultry by highly virulent NDV are notifiable to the OIE, provided the causative APMV-1 strain meets one of the following criteria for virulence (6): “(i) The virus has an ICPI in day-old chicks (Gallus gallus) of 0.7 or greater. (ii) Multiple basic amino acids have been demonstrated in the virus at the C terminus of the F2 protein and phenylalanine at residue 117, which is the N terminus of the F1 protein. The term ‘multiple basic amino acids’ refers to at least 3 arginine or lysine residues between residues 113 and 116. Failure to demonstrate the characteristic pattern of amino acid residues as described above would require characterization of the isolated virus by an ICPI test.” As a result, the direct or indirect determination of the amino acid sequence at the F0 protein cleavage site allows confirmation of virus virulence, but not the lack of virulence.

Comparative sequence analysis of the HN and F protein genes from virulent and avirulent NDV isolates revealed that pathogenicity is influenced by the length of the HN protein and the amino acid sequence/composition of the proteolytic cleavage site of the F0 protein precursor encoded by the F gene (38). The cleavage of the precursor protein is a prerequisite for viral infection, as virus particles are activated only if F0 is enzymatically cleaved into functional F1 and F2 peptides, which enables the viral and host cell membranes to fuse. Based on the data obtained by sequence analysis, NDV strains that are virulent for chickens contain at least three basic residues (lysine and arginine [K and R]) between positions 113 and 116 at the C terminus of the F2 protein (cleavage site motif 112R/K-R-Q-R/K-R-F117) and phenylalanine (F) at the N terminus of the F1 protein (residue 117). As a result, the precursor protein of virulent isolates is more susceptible to cleavage by the ubiquitous proteases present in different tissues of the host (13). Strains of low virulence have fewer basic amino acid residues at the C terminus of the F2 protein and leucine (L) at position 117 (cleavage site motif 112G/E-K/R-Q-G/E-R-L117), and trypsin-like proteases are required to cleave their precursor. In summary, the virulence of NDV is highly dependent on the cleavability of the fusion glycoprotein precursor (F0) by cellular proteases of the host (3). Another important conclusion from the sequence analysis is that the mesogenic and velogenic viruses cannot be differentiated on the basis of the nucleotide sequence of the F0 proteolytic cleavage site, and the same is true for asymptomatic enteric and lentogenic viruses (40).

Currently, the isolation of the causative virus using embryonated chicken eggs and its characterization by hemagglutination activity (HA)/hemagglutination inhibition/in vivo pathogenicity assays is the standard protocol to confirm ND cases (6). Although conventional microbiological methods are sensitive and specific, it takes considerable time (4 to 7 days) to make a diagnosis; therefore, PCR-based detection and pathotyping assays have been developed to support/substitute for these traditional diagnostic tools and to replace animal experiments (4, 15, 29, 37, 43).

The discrimination of avirulent (e.g., vaccine viruses) and virulent NDV strains is very important in regions practicing vaccination as a control measure against ND, especially in the context of the differential diagnosis of avian influenza (6). In this paper, we describe the construction of a real-time reverse transcription-PCR (RRT-PCR) assay using fluorescent minor groove binder (MGB) TaqMan probes to pathotype NDV strains. (R)RT-PCR methods provide high sensitivity and opportunity for quantitative measurements, and by omitting post-PCR steps, they reduce the risk of cross-contamination and the time and material needed to make a diagnosis (9). The specificity of the method was tested with heterologous (viral/bacterial) avian pathogens, archive isolates, and clinical samples, and it was found to be appropriate for the detection and the rapid, accurate pathotyping of NDV.

MATERIALS AND METHODS

Primer design and PCR.

Nucleotide sequences of NDV F genes available in GenBank were aligned by using the computer program CLC Free Workbench version 4.5.1 (CLC bio A/S, Aarhus, Denmark). Degenerate oligonucleotide amplification primers and custom TaqMan MGB probes targeting the fusion protein cleavage site (F0) of lentogenic and velogenic/mesogenic NDV strains were designed by Primer Express version 3.0 (Applied Biosystems, Foster City, CA). The specificity of primers/probes was tested by using a GenBank BLAST search to exclude the possibility of false-positive results with heterologous sequences. Unlabeled amplification primers and custom TaqMan MGB probes carrying 6-carboxyfluorescein reporter dye at the 5′ end and nonfluorescent MGB quencher at the 3′ end were purchased from Biomi (Gödöllő, Hungary) and Applied Biosystems, respectively. Primer/probe sequences used in the pathotyping assay and their annealing sites in different NDV strains are shown in Tables 1 and 2, respectively. The PCR mixture consisted of 8.3 μl PCR grade H2O (Life Technologies, Gaithersburg, MD), 2 μl AmpliTaq Gold 10× PCR buffer, 3.2 μl 25 mM MgCl2, 0.1 μl AmpliTaq Gold polymerase (5 U/μl; Applied Biosystems), 0.4 μl 10 mM deoxynucleotide triphosphate blend, 0.5 μl 5-mg/ml bovine serum albumin (Promega, Madison, WI), 1.5 μl of each forward and reverse amplification primer (10 pmol/μl; Biomi), 1.5 μl of TaqMan MGB probe (10 pmol/μl; Applied Biosystems), and 1 μl template cDNA/DNA. When two primers/probes were used in combination (e.g., FT_NDV_VR2 and -3), 0.75 μl of each component was added to the reaction mixture. For real-time PCR amplification, the cycling program recommended by the manufacturer of the TaqMan MGB probes (Applied Biosystems) was used: 95°C for 10 min (hot start), followed by 45 cycles of 95°C for 15 s and 60°C for 60 s. Fluorescence data were collected in the primer elongation phase at 60°C. RRT-PCR was performed in the following thermocyclers: iCycler, iQ5 (Bio-Rad, Hercules, CA), and ABI 7500 (Applied Biosystems).

TABLE 1.

Oligonucleotide primers/probes used for RRT-PCR detection of lentogenic and velogenic/mesogenic NDV F gene sequences

NDV specificity Primer Sequence (5′→3′)a Position (bp)b Amplicon size (bp)
Lentogens FT_NDV_LF3 TCC GBA GGA TAC AAG AGT CYG TGA CC 4839-4864 85
FT_NDV_LF4 TCC GBA GGA TAC AAG AGT CYG TGA CT
FT_NDV_LR2 AGA GCY ACA CCG CCA ATA AT 4923-4904
FT_NDV_LR3 AGA GCY ACA CCA CCG ATA AT
FT_NDV_Lprobe2 CAG GGR CGC CTT ATA 4883-4897
Velogens/mesogens FT_NDV_VF1 GAY TCY ATC CGY AGG ATA CAA GRG TC 4832-4857 99
FT_NDV_VR2 AAC CCC AAG AGC TAC ACY RCC 4930-4910
FT_NDV_VR3 GAC CCC AAG AGC TAC ACY RCC
FT_NDV_Vprobe1 AAR CGT YTC TGY CTC C 4893-4878
FT_NDV_Vprobe2 AGA RAC GCT TTR TAG GTG C 4884-4902
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a

Y = T or C; R = G or A; B = C, G, or T.

b

Nucleotide positions correspond to the NDV B1 complete genome (GenBank accession number AF309418).

TABLE 2.

Primer/probe annealing and F0 cleavage sites of lentogenic and velogenic/mesogenic NDV strains used in the specificity testsa

NDV strain Annealing site
F0 cleavage siteb (amino acid positions 112-117)
Forward primer TaqMan MGB probeb Reverse primer
Lentogen-specific primer or probe TCCGBAGGATACAAGAGTCYGTGACCc CAGGGRCGC↓CTTATAe ATTATTGGCGGTGTRGCTCTf
.........................Td .....C..T...........g
Lentogenic strain
    HK-99/75 GATTCTA....T..............T...... GGAAA.....A...↓..C...GGCGC .....C..T..C.CA.....CGGGGTT GKQGR↓L
    HK-138/80 GATTCCA....T..............T...... GGAAA.....A..T↓......GGCGC .....C..T.....A.....CGGGGTT GKQGR↓L
    Ulster 2/C GATTCTA.T..T..............T.....T GGAAA.....A...↓......GGCGC .....C.......CA.....CGGGGTT GKQGR↓L
    PHY-LMV42 GATTCTA....T..............T...... GGAAA.....A...↓......GGCGC .....C..T.....A.....CGGGGTT GKQGR↓L
    Queensland V4 GATTCTA....T..............T...... GGAAA.....A..T↓......GGCGC .....C..T.....A.....CGGGGTT GKQGR↓L
    CA-12/63 GATTCTA.T..T..............T...... AGAAA.....G...↓......GGTGC ..............A.....TGGGGTT EKQGR↓L
    B1 GACTCTA....T..............T.....T GGAGA.....G...↓......GGCGC ..............G.....TGGGGTT GRQGR↓L
    LaSota GACTCTA....T..............T.....T GGAGA.....G...↓......GGCGC ..............G.....TGGGGTT GRQGR↓L
    HU/ck/146-V/95 GACTCTA.T..T..............T.....T GGAGA.....G...↓......GGCGC ..............G.....TGGGGTT GRQGR↓L
Velogen/mesogen-specific primer or probe GAYTCYATCCGYAGGATACAAGRGTCh GGAGRCAGARACGY↓TTi     GGYRGTGTAGCTCTTGGGGTTk
AGARACGC↓TTTRTAGGTGCj     ....................Cl
Velogenic/mesogenic strain
    BG-13/69 ..C..T.....T..........A...TGTAACT ....A....A...A↓...A....C.. ATTATT..CG....G............ RRQKR↓F
    UK-Eaw/62 ..C..T.....T..........A...CGTAACT ....A....A...C↓...A....C.. ATTATT..CG....G..C......... RRQKR↓F
    Beaudette C ..T..T.....T..........G...TGTAACT ....A....A...C↓...A....C.. ATTATT..CG....G............ RRQKR↓F
    KR-1/49 ..T..T.....C..........A...TGTGACT ....A....A...C↓..CG....... ATTATT..CA..........C...... RRQRR↓F
    Mukteswar ..T..T.....C..........A...TGTGACT ....A....G...C↓...A....... ATTATT..CA..........A...... RRQRR↓F
    H/Ph ..T..T.....C..........A...TGTGACT ....A....G...C↓...A....... ATTATT..CA..........A...... RRQRR↓F
    Herts'33/L ..T..T..T..T..........A...AGTGACT ....A....G...C↓...A....C.. GTAATC..TG.............A... RRQRR↓F
    BG-60/81 ..T..T.....C..A.......A...TGTGACT ....A....G...C↓...A....... ATTATC..CA.......C.....A... RRQRR↓F
    IT-7/60 ..T..C.....C..........A...TGTGACT ....A....G...C↓...A....... ATTATC..CA..A.............. RRQRR↓F
    MA-307/77 ..T..T.....C........G.A...TGTGACT ....A....G...C↓...G....C.. ATTATT..CA....G........A... RRQRR↓F
    BG-25/78 ..C..C..T..A..........G...TGCGACT ....A....A...C↓...G....... ATTATT..CA................. RRQKR↓F
    HU-7/72 ..C..C.....A..........G...TGCGACT ....A....A...C↓...G....... ATTATC..CA................. RRQKR↓F
    KR-2/84 ..C..C.....C..........G...TGTGACT ....A....A...C↓...G....... ATTATC..CA................. RRQKR↓F
    PE-8/85 ..C..C.....C..A.......G...TGCGACC ....A....A...C↓...T....... ATTATC..CA................. RRQKR↓F
    BG-29/86 ..T..C.....C..........G...TGTGTCT ....A....A...C↓...A....... ATTATC..AA................C RRQKR↓F
    GR-12/68 ..T..C.....C..........G...TGTGTCC A...A....A...C↓...A....... ATTATC..CA................C KRQKR↓F
    IQ-218/78 ..T..C.....C..........G...TGTGTCC ....G....A...C↓...A....... ATTATC..CA................C RRQKR↓F
    Israel 70 ..T..C.....C..........G...TGTGTCT ....A....A...C↓...A....... ATTATC..CA................C RRQKR↓F
    KR-5/99 ..T..C.....C.A........G...CGTGTCA ....A.GA.A...C↓...A....... ATTATC..TA..........C.....C RRRKR↓F
    CA-48/91 ..T..C.....C.A........G...TGTGTCC A...G....A...C↓...A....... ATTATC..CA................C KRQKR↓F
    HU-238/84 ..T..C.....C..........G...TGTGTCC ....G....A...C↓...A....... ATTATC..CA................C RRQKR↓F
    IT-227/82 ..T..C.....C..........G...TGTGTCC ....G....A...C↓...A....... ATTATC..CA................C GRQKR↓F
    BE-14/93 ..T..C.....C.A...C....G...TGTGTCC ....A....A...C↓...A....... GTTATT..CA................. RRQKR↓F
    BG-31/96 ..T..T.....T..........G...TGTGTCC ....A....G...T↓...A....... GTTATT..CA.......C......... RRQRR↓F
    DE-82/94 ..T..C.....C.A...C....GT..TGTGTCC ....A....A...C↓...A....... GTTATT..CA................. RRQKR↓F
    ID-1/88 ..T..C.....C.A...C....G...TGTGGCC ....A....A...C↓...A....... GTTATT..CA.............A... RRQKR↓F
    TR-2/96 ..T..T..T..T..........G...TGTGTCC ....A....G...T↓...A....... ATTATC..CA................. RRQRR↓F
    IT-147/94 ..T..C.....C..........G...TGTGACT ....A....AG..C↓...A....... GTTATC..TA.......C..C..A... RRQKR↓F
    JP-382/84 ..T..C.....C..........G...TGTGACT ....A....AG..C↓...A....... ATTATC..GA.......C.....A... RRQKR↓F
    ZA-16/90 ..T..T.....C..........G...TGTAACT .A..A.G..A...C↓...A....... ATTATC..TA.C.....C.....A... RRRKR↓F
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a

Nucleotide sequences are given in the 5′→3′ direction (for nucleotide positions, see Table 1).

b

The arrows represent the F0 cleavage sites. The nucleotides corresponding to the F0 cleavage site motif (amino acid residues 112 to 117) are in italics.

c

Primer FT_NDV_LF3.

d

Primer FT_NDV_LF4.

e

Probe FT_NDV_Lprobe2.

f

Reverse complement of primer FT_NDV_LR2.

g

Reverse complement of primer FT_NDV_LR3.

h

Primer FT_NDF_VF1.

i

Reverse complement of probe FT_NDV_Vprobe1.

j

Probe FT_NDV_Vprobe2.

k

Reverse complement of primer FT_NDV_VR2.

l

Reverse complement of primer FT_NDV_VR3.

Nucleic acid extraction and cDNA synthesis.

Nucleic acid from APMV-1 and heterologous RNA virus samples was extracted by using the QIAamp viral RNA Mini kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. Lyophilized vaccine stock reconstituted in sterile distilled water (NDV vaccine strains), allantoic fluid (all other NDV strains), and allantoic fluid/tissue culture (other RNA viruses) were the sample sources for RNA purification. The RNA concentrations of the preparations used in the NDV specificity test (Table 3) were quantitated spectrophotometrically by using a Nanodrop ND-1000 spectrophotometer. Mean values were calculated from triplicate measurements, and the template RNA concentration was adjusted to 65 to 85 ng/μl by adding diethylpyrocarbonate-treated sterile nuclease-free water to dilute the RNA samples when necessary. The RT of RNA to cDNA was performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA), pd(N)6 random hexamer 5′-phosphate, and RNAguard (Amersham, Piscataway, NJ) according to the manufacturers' instructions. For extraction of DNA from heterologous viruses and bacteria, the Chelex-100 method was applied as described by Fenicia et al. (22). For the purification of RNA from experimentally infected animals, see below.

TABLE 3.

NDV strains tested by the NDV-pathotyping RRT-PCR and their results in the specificity assay

NDV straina Host GenBank accession no. Genotype Virulenceb RRT-PCR specific forc:
Lentogens
Velogens/mesogens
Test result CT ± SD Test result CT ± SD
HK-99/75 Duck EU604269 I L + 34.0 ± 1.1 − NA
HK-138/80 Duck EU604270 I L + 18.9 ± 0.8 − NA
Ulster 2/C Chicken AY562991 I L + 23.6 ± 0.2 − NA
PHY-LMV42 Chicken DQ097394 I L + 20.7 ± 0.5 − NA
Queensland V4 Chicken AF217084 I L + 16.8 ± 0.3 − NA
CA-12/63 Turkey EU604268 II L + 14.7 ± 0.2 − NA
BG-13/69 Chicken EU604248 II V − NA + 25.8 ± 1.0
UK-Eaw/62 Chicken EU604260 II V − NA + 18.4 ± 0.4
B1 Chicken AF309418 II L + 16.4 ± 0.2 − NA
LaSota Chicken AJ629062 II L + 15.5 ± 0.4 − NA
HU/ck/146-V/95 Chicken EU604271 II L + 20.8 ± 0.7 − NA
Beaudette C Chicken X04719 II M − NA + 16.1 ± 0.3
KR-1/49 Chicken EU604814 III V − NA + 16.9 ± 0.4
Mukteswar Chicken EF201805 III M − NA + 13.8 ± 0.3
H/Ph Chicken FJ687488 III M − NA + 14.5 ± 0.2
Herts'33/L Chicken FJ687487 H33 W V − NA + 21.5 ± 0.4
BG-60/81 Chicken EU604266 IV V − NA + 20.7 ± 0.5
IT-7/60 Chicken EU604256 IV V − NA + 15.7 ± 0.5
MA-307/77 Chicken EU604259 IV V − NA + 21.3 ± 0.3
BG-25/78 Chicken EU604249 V V − NA + 17.0 ± 0.3
HU-7/72 Chicken EU604252 V V − NA + 13.3 ± 0.1
KR-2/84 Chicken EU604258 V V − NA + 12.9 ± 0.5
PE-8/85 Chicken EU604263 V V − NA + 15.5 ± 0.3
BG-29/86 Chicken EU604267 VI V − NA + 15.2 ± 0.7
GR-12/68 Chicken EU604251 VI V − NA + 13.2 ± 0.3
IQ-218/78 Pigeon EU604254 VI M − NA + 13.6 ± 0.2
Israel 70 Chicken EU604255 VI V − NA + 13.5 ± 0.3
KR-5/99 Chicken EU665683 VI V − NA + 17.1 ± 0.1
CA-48/91 Pigeon FJ687489 VI M − NA + 12.6 ± 0.3
HU-238/84 Pigeon FJ687486 VI M − NA + 15.3 ± 0.2
IT-227/82 Pigeon AJ880277 VI M − NA + 15.9 ± 0.4
BE-14/93 Chicken EU604247 VII V − NA + 15.2 ± 0.2
BG-31/96 Chicken EU604250 VII V − NA + 15.8 ± 0.1
DE-82/94 Chicken EU604265 VII V − NA + 25.4 ± 0.2
ID-1/88 Cockatoo EU604253 VII V − NA + 13.9 ± 0.2
TR-2/96 Chicken EU604264 VII V − NA + 23.4 ± 0.3
IT-147/94 Turkey EU604262 VIII V − NA + 28.8 ± 0.1
JP-382/84 Chicken EU604257 VIII V − NA + 29.0 ± 0.1
ZA-16/90 Chicken EU604261 VIII V − NA + 30.8 ± 0.3
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a

For APMV-1 isolates, the year/country of isolation are indicated in the designation of the virus (e.g., ZA-16/90: country of origin, South Africa; year of isolation, 1990; see also ISO 3166 two-letter country code standard for details [http://www.iso.org]).

b

L, lentogenic; M, mesogenic; V, velogenic.

c

NA, not applicable; +, postive; −, negative.

PCR specificity and sensitivity assay.

The performance of the PCR specificity and sensitivity assay was assessed by using RNA/DNA extracted from NDV strains and 24 heterologous avian pathogens (Tables 3 and 4). Nucleic acid samples were tested as described above. The detection limit of the pathotyping PCR assay was determined using the target copy number and virus titer (50% egg infective dose [EID50]) as reference units. (i) Serial 10-fold dilution series (102 to 1010×) were prepared from pKR5-FHN/pCR4-F plasmid constructs carrying the F gene from velogenic (KR-5/99)/lentogenic (PHY-LMV42) NDV strains, respectively. One microliter of plasmid DNA from each dilution was added to the PCR as a template. (ii) Serial 10-fold dilutions (101 to 108×) of NDV stock solutions with known EID50 values (HU/ck/2294-BI/04, lentogenic, titer = 108.5 EID50/ml; Mukteswar, mesogenic, titer = 107.2 EID50/ml) were prepared using sterile H2O. RNA was extracted from each dilution and transcribed as described above, and cDNA was added to the PCR mixture as a template. In the real-time PCR experiments, threshold cycle (CT) values were determined automatically by the softwares of the iCycler/iQ5 (Bio-Rad) and ABI 7500 (Applied Biosystems) thermocyclers. The mean CT and standard deviation values (Table 3) were calculated from triplicate reactions.

TABLE 4.

Heterologous avian pathogens tested by the NDV-pathotyping RRT-PCR and their results in the specificity assay

Heterologous virus/bacterium strainb Test resulta of RRT-PCR specific for:
Lentogenic NDV Velogenic/mesogenic NDV
Avian influenza virus − −
Avian metapneumovirus − −
Avian polyoma virus − −
Avian reovirus − −
Chicken anemia virus − −
Derzsy's disease virus − −
Infectious bronchitis virus − −
Infectious laryngotracheitis virus − −
M. gallisepticum − −
Mycoplasma iowae − −
Mycoplasma meleagridis − −
Mycoplasma synoviae − −
Ornithobacterium rhinotracheale − −
Pasteurella multocida − −
APMV-2/robin/Hiddensee/19 − −
APMV-2/chicken/California, Yucaipa/56 − −
APMV-3/parakeet/NL/449/75 − −
APMV-4/duck/HK/D3/75 − −
APMV-6/duck/HK/199/77 − −
APMV-7/dove/Tennessee/4/75 − −
Psittacid herpesvirus 1 − −
Riemerella anatipestifer − −
Turkey adenovirus − −
Turkey astrovirus − −
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a

−, negative.

b

HK, Hong Kong; NL, The Netherlands.

Plasmid standards.

Plasmid constructs carrying the F gene from the lentogenic strain PHY-LMV42 (pCR4-F; vector, pCR4-TOPO [Invitrogen, Carlsbad, CA]) and the velogenic isolate KR-5/99 (pKR5-FHN; vector, pCR-XL-TOPO [Invitrogen]), respectively, were kindly supplied by A. Czeglédi and P. Élő (Veterinary Medical Research Institute of the Hungarian Academy of Sciences [VMRI], Budapest, Hungary). Plasmid preparations were quantified spectrophotometrically by using a Nanodrop ND-1000 spectrophotometer, and plasmid copy numbers were calculated. Dilutions of the plasmids were used in real-time PCRs to prepare standard curves.

Isolates and sequence data.

The NDV strains and heterologous avian pathogens used in the specificity tests are listed in Tables 3 and 4. In the case of NDV isolates, the virulence properties, genotypes, countries/hosts of origin, and years of isolation are shown. APMV isolates (with the exception of the NDVs listed below) were kindly provided by B. Lomniczi (VMRI, Budapest, Hungary). Previously described NDVs (8, 16, 17, 18, 24, 25, 32) are shown with the original designation; only the country code was modified according to the International Standards Organization 3166 two-letter standard (e.g., B-14/93 was modified to BE-14/93). Genotype and pathotype grouping was carried out by B. Lomniczi (VMRI). The vaccine NDV strains B1, LaSota (Phylavac), PHY-LMV42 (Vitapest), Queensland V4, H/Ph (vaccine H), and Mukteswar were obtained from Ceva-Phylaxia (Budapest, Hungary). The genotype and pathotype classifications of vaccine strains were adopted from Ballagi-Pordány et al. (8). Other virus and bacterial field isolates, including heterologous pathogens, were collected from clinical samples in the Microbiology Department of the Central Agricultural Office, Veterinary Diagnostic Directorate (Debrecen, Hungary). Of the six NDV isolates collected in the Central Agricultural Office, Veterinary Diagnostic Directorate, five were identical to vaccine strains in the region amplified by primers A and B (29): three LaSota (HU/ck/39-V/85, HU/ck/1377-V/86, and HU/ck/20-V/87), one PHY-LMV42 (HU/ck/2294-BI/04), and one Queensland V4 (HU/ck/2792-V/98) strain were recovered from clinical samples. To avoid redundancies, only the results of the LaSota progeny isolate HU/ck/146-V/95, which carries one point mutation in the primer annealing site (Table 2), are presented in Table 3. After propagation in embryonated chicken egg, isolate HU/ck/2294-BI/04 was titrated and used in the determination of the detection limit. Prior to their application in specificity assays, bacterial and viral nucleic acid samples were tested and proved to be positive by the respective diagnostic methods, including PCR detection (data not shown). NDV samples were first identified by propagation in embryonated chicken eggs and an HA test, according to the OIE manuals (6); then, the results were confirmed by sequencing a 325-bp-long region of the F gene with primers A and B (29). The QIAquick PCR purification kit (Qiagen) was used for the purification of amplified DNA fragments according to the manufacturer's instructions. Double-stranded sequencing reactions were performed by using the BigDye Terminator Cycle Sequencing kit v3.1 (Applied Biosystems) and primers A and B (29). Samples were analyzed in an ABI3100 automated sequencer (Biomi). Virus titer values were determined as described previously (21).

Detection of NDV in organ and swab samples from experimentally infected chickens.

Specific-pathogen-free chicks were vaccinated intraocularly at 1 day of age with one dose of Cevac Vitapest L containing the NDV strain PHY-LMV42 (CEVA-Phylaxia). Organ (brain, conjunctiva, trachea, lung, and cecal tonsil) and swab (oral and cloacal) samples were taken at days 2, 5, and 9 postvaccination (p.v.). The organs were weighed and homogenized manually with sterile quartz sand in sterile phosphate-buffered saline containing 0.01% gentamicin sulfate, 0.01% colistin sulfate, and 0.005% norfloxacin (Sigma-Aldrich, St. Louis, MO) to make a 1:10 (wt/vol) dilution. Oral and cloacal swab samples were suspended in 1 ml sterile phosphate-buffered saline containing the antibiotics mentioned above. RNA was extracted from the organ and swab samples by using TRI reagent LS (Sigma-Aldrich) as recommended by the manufacturer. RNA was resuspended in 25 μl diethylpyrocarbonate-treated sterile nuclease-free water and was stored at −80°C. RT was performed as described above, and cDNA was added to the lentogenic NDV-specific real-time PCR assay as a template. The CT was determined automatically by the software of the thermocycler, and mean CT/standard deviation values (Table 5) were calculated from triplicate reactions. In the control reaction, the same amount of cDNA template was added to the gel-based assay using primers A and B (29).

TABLE 5.

Results of lentogen-specific TaqMan MGB and gel-based PCR assays for organ and swab samples from experimentally infected chickens

Samplea Test result of PCR
Lentogen-specific TaqMan MGB assayb
Gel-based assay using NDV primers A + Bc
Result CT ± SD
Sampling at 2 days p.v.
    Brain − NA Negative
    Conjunctiva ++ 27.8 ± 0.7 Positive
    Trachea + 33.5 ± 0.6 Negative
    Cecal tonsil − NA Negative
    Lung + 31.7 ± 0.2 Negative
    Oral swab + 30.3 ± 0.2 Negative
    Cloacal swab − NA Negative
Sampling at 5 days p.v.
    Brain + 38.0 ± 2.0 Negative
    Conjunctiva ++ 27.4 ± 0.5 Positive
    Trachea ++ 26.4 ± 0.4 Positive
    Cecal tonsil − NA Negative
    Lung + 31.7 ± 0.3 Negative
    Oral swab + 33.0 ± 0.4 Negative
    Cloacal swab + 31.7 ± 0.2 Negative
Sampling at 9 days p.v.
    Brain − NA Negative
    Conjunctiva + 35.9 ± 0.5 Negative
    Trachea +/− 42.9 ± 0.8 Negative
    Cecal tonsil +/− 43.1 ± 0.7 Negative
    Lung − NA Negative
    Oral swab − NA Negative
    Cloacal swab ++ 28.2 ± 0.2 Positive
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a

Samples were taken 2, 5, and 9 days p.v. of chicks at 1 day of age with NDV strain PHY-LMV42 (Vitapest).

b

RRT-PCR test result: strong positive (++), CT < 30; positive (+), 30 < CT < 40; weak positive (+/−), CT > 40; negative (−), at least two negative results in triplicate reactions (CT is not applicable). NA, not applicable.

c

See reference 29.

Detection of NDV in stool samples from experimentally infected chickens.

A breeder flock was vaccinated with Avinew (Merial, Lyon, France) at 1 day and with TAD ND vac HB1 (Lohmann, Cuxhaven, Germany) at 21 days of age. Vaccines contained VG/GA and Hitchner B1 live NDV strains, respectively. Two weeks after vaccination (day 14 p.v.), stool specimens were collected and pooled according to a European Communities directive (regulation no. 1003/2005) (14). To 0.2 g pooled feces, 0.5 ml sterile H2O was added, and then samples were thoroughly vortexed and centrifuged (2 min; 8,000 rpm; room temperature). Viral RNA was extracted from the supernatant by using the QIAamp Viral RNA Mini kit (Qiagen) as recommended by the manufacturer. RT was performed as described above, and cDNA was added to the lentogenic NDV-specific real-time PCR mixture as a template. The results were confirmed by a gel-based PCR assay using universal and pathotype-specific primer pairs (29). All PCR experiments were carried out in duplicate.

Nucleotide sequence accession numbers.

The partial nucleotide sequences of 31 NDV strains have been deposited in GenBank (accession numbers EU604247 to EU604271, EU604814, EU665683, and FJ687486 to FJ687489).

RESULTS

Specificity of the TaqMan MGB RRT-PCR assay.

NDV isolates representing genotypes I to VIII (8, 17) and clinical isolates were subjected to the TaqMan MGB RRT-PCR tests. The primer/probe combination FT_NDV_LF3 and -4, FT_NDV_LR2 and -3, and FT_NDV_Lprobe2 amplified an 85-bp fragment of almost all NDV strains (data not shown), but fluorescent signal was detected only in the nine lentogenic samples (Table 3). The five clinical isolates that shared 100% homology with the vaccine strains (see Materials and Methods) and HU/ck/146-V/95, carrying a single point mutation in the primer annealing site, also tested positive in the reaction (only the result for the last isolate is shown in Table 3). Isolates that were found positive in the assay belonged to genotypes I and II and contained seven different primer/probe annealing site combinations. On the nucleic acid level, a total of four forward primer, four probe, and five reverse primer annealing site variants were investigated in the experiment. On the amino acid level, the following lentogenic F0 cleavage site (↓) motifs were represented in the tests (amino acid positions 112 to 117): G-K-Q-G-R↓L, E-K-Q-G-R↓L, and G-R-Q-G-R↓L (Tables 2 and 3). Of the nine lentogenic NDV strains tested, six were complementary to one of the degenerate probes and three had one mismatch in the probe annealing region. A significantly high CT value (34.0) was observed in the latter group for HK-99/75, which carried two additional mismatches in the reverse primer annealing site, while in the other cases, the noncomplementary base pairs of the probe annealing sites had no apparent effect on PCR efficiency (e.g., Queensland V4 had a lower CT value than PHY-LMV4). Strains with identical nucleotide sequences in the primer/probe annealing sites (Queensland V4 and HK-138/80 from genotype I; B1 and LaSota from genotype II) resulted in comparable CT values. Based on the available data, no direct correlation could be detected between the number and positions of noncomplementary base pairs and PCR efficiency.

In the reaction designed to detect the F0 region from virulent viruses, amplification primers FT_NDV_VF1/FT_ NDV_VR2 and -3 yielded a 99-bp fragment in all NDV strains (data not shown), but fluorescent signal was observed only in the cases of velogens and mesogens classified in genotypes II to VIII (Table 3). Virulent strains were more variable in the cleavage site region of the fusion protein precursor than lentogens: 27 of the 30 isolates carried unique primer/probe annealing site combinations. On the nucleic acid level, 17 forward primer, 16 probe, and 17 reverse primer annealing regions were represented in the virulent samples, while on the amino acid level, five cleavage site motifs (R-R-Q-K-R↓F, R-R-Q-R-R↓F, and K-R-Q-K-R↓F, as well as “nonstandard” motifs R-R-R-K-R↓F in isolates ZA-16/90 and KR-5/99 and G-R-Q-K-R↓F in isolate IT-227/82) were represented. Of the 30 velogenic and mesogenic strains, three isolates (BG-13/69, KR-5/99, and ZA-16/90) contained two mismatches in the probe annealing sites. The highest CT value was measured in strain ZA-16/90, which contained 1 and 2 bp noncomplementary to fluorescent probes FT_NDV_Vprobe2 and FT_NDV_Vprobe1, respectively. In isolate KR-5/99, the two mismatches affected both TaqMan MGB probes, but the efficiency of the reaction was not reduced significantly, as was shown by the CT value in Table 3 and the results of the sensitivity test, in which the detection limit was determined for velogens/mesogens in terms of the target copy number. In the case of the third member of the group, BG-13/69, which carried one additional mismatch in the reverse primer binding site besides the noncomplementary base pairs in the probe annealing site, the CT value was not significantly high, but the relative fluorescence signal (measured in relative fluorescence units) was very weak. The CT values of the viruses containing a single mismatch in the probe annealing region varied over a wide range (12.6 to 29.0). Although some correlations were observed—notably, that isolates containing the same primer/probe binding sites had similar CT values (e.g., Mukteswar and H/Ph from genotype III and IQ-218/78, HU-238/84, and IT-227/82 from genotype VI), and that in strains with identical probe annealing sites, the more mismatches were in the primer annealing sites, the higher the CT value measured in the real-time assay (e.g., H/Ph, IT-7/60, and BG-60/81 from genotypes III and IV and KR-2/84, HU-7/72, and BG-25/78 from genotype V)—the exact relationship between the number and locations of mismatches in the primer/probe annealing sites and CT values could not be determined (e.g., Israel 70, BG-29/86, BE-14/93, DE-82/94, and ID-1/88 from genotypes VI and VII).

In the control experiments, a panel of heterologous avian pathogens was tested by the NDV-pathotyping reactions. No false-positive results were observed with the 24 viral/bacterial strains in the RRT-PCR assay specific for lentogenic and velogenic/mesogenic NDVs (Table 4).

Sensitivity of the TaqMan MGB RRT-PCR assay.

The analytical sensitivity of the pathotyping assay was evaluated by using 10-fold serial dilutions of (i) plasmid constructs containing the cloned F gene from lentogenic (PHY-LMV42) and velogenic (KR-5/99) isolates and (ii) lentogenic and mesogenic NDV stock solutions with known EID50 values. In the first case, when plasmid DNA was added to the reaction mixture as a template, the detection limit of the PCR specific for lentogens was determined to be approximately 10 copies of the target molecule per reaction. Plotting the log of the template concentration (copy number) versus the CT generated a standard curve, which had a correlation coefficient (R2) value of 0.998. The quantification graph had a wide dynamic range over 8 orders of magnitude (1.0 × 108 to 10 starting plasmid copy number), and the calculated PCR efficiency was 81.5% (slope, −3.862). In the case of the PCR specific for velogens and mesogens, the detection limit was approximately 20 copies per reaction, the quantification was linear over a range of 2.4 × 107 to 24 starting plasmid copy number, the calculated PCR efficiency was 101% (slope, −3.299), and the correlation coefficient was 0.999.

In the second set of experiments, cDNA templates were prepared from dilution series of lentogenic and mesogenic NDV stock solutions and used as templates in the reaction. In the case of the lentogenic strain HU/ck/2294-BI/04, which was identical in the region amplified by primers A and B to the lentogenic vaccine strain PHY-LMV42, the detection limit of the PCR specific for lentogens was approximately 10 EID50/ml (original titer of virus, 108.5 EID50/ml). The PCR efficiency was 89% (slope, −3.619), and the correlation coefficient was calculated as 0.998. The mesogenic strain Mukteswar (original titer of virus, 107.2 EID50/ml) was used as a template in the PCR specific for velogens/mesogens to measure the limit of detection, which was determined to be approximately 10−1.3 EID50/ml. The PCR efficiency was 97% (slope, −3.395), and the correlation coefficient was calculated as 0.999. The quantification curves were linear over 5 orders of magnitude in both reactions.

Detection of NDV in organ and swab samples from experimentally infected chickens.

In order to evaluate the clinical applicability of the assay, specific-pathogen-free chicks were vaccinated at 1 day of age with NDV strain PHY-LMV42, and then samples were taken at days 2, 5, and 9 p.v. and tested by the TaqMan MGB RRT-PCR specific for lentogens (Table 5) and a gel-based PCR using primers A and B (29). PCR was performed on RNA extracted from brain, conjunctiva, trachea, lung, and cecal tonsil samples and oral and cloacal swab samples. In the real-time PCR, a strong fluorescent signal (CT < 30) was measured in the samples prepared from conjunctiva at 2 days p.v., from the conjunctiva and trachea at 5 days p.v., and from the cloacal swab at 9 days p.v. A weaker fluorescent signal (30 < CT < 40) was observed in the following samples: trachea, lung, and oral swab at 2 days p.v.; brain, lung, and oral and cloacal swabs at 5 days p.v.; and conjunctiva at 9 days p.v. Trachea and cecal tonsil samples at 9 days p.v. gave a very weak positive signal (40 < CT), while other samples were found negative in the real-time assay: either no fluorescent signal was detected (cecal tonsil at 5 days p.v. and oral swab at 9 days p.v.), or mixed negative and weak positive test results were obtained in the triplicate reactions (brain, cecal tonsil, and cloacal swab at 2 days p.v. and brain and lung at 9 days p.v.).

The real-time PCR test results for the samples that gave strong fluorescent signals (conjunctiva at 2 and 5 days p.v., trachea at 5 days p.v., and cloacal swab at 9 days p.v.) were confirmed when the expected fragment was amplified from these samples in the gel-based control PCR. All other samples were negative in the assay using the primer pair A and B.

Detection of NDV in stool samples from experimentally infected chickens.

Stool samples collected from a chicken flock previously inoculated with NDV vaccine strains VG/GA and Hitchner B1 were tested by (i) gel-based PCR for the presence of NDV (21), (ii) gel-based pathotyping PCR (29), and (iii) RRT-PCR specific for lentogens. Amplification products and fluorescent signals were detected in the samples originating from vaccinated birds, while in the control reactions with samples collected from nonvaccinated control flocks and no template controls, no amplification product was observed.

DISCUSSION

In this report, we described the development of a novel pathotyping real-time PCR assay capable of detecting and differentiating NDV isolates by using TaqMan MGB probes. Similarly to previously published molecular-biology-based detection/pathotyping methods applying digoxigenin (35) or radioactively labeled hybridization probes (27), light upon extension (7), SYBR green (37) technology, and fluorogenic hydrolysis probes (4, 43), our assay also aimed to provide a labor- and time-saving but reliable tool for rapid diagnosis of ND that gives results days ahead of the conventional immunology-based tests (hemagglutination inhibition/HA). The assay utilizes a fluorescent 5′-nuclease PCR technology for this purpose, which has the potential to be used for quantitative measurements and eliminates postamplification procedures and thus reduces the risk of contamination and handling errors. The viability of the TaqMan platform has been proven by its numerous applications in PCR-based assays, e.g., to detect OIE-notifiable avian pathogens: avian influenza A/H5/H7 virus (2, 20, 33, 41), infectious bronchitis virus (10), infectious laryngotracheitis virus (11), Mycoplasma gallisepticum (12), and infectious bursal disease virus (34). Applying MGB quenchers in the 3′ ends of TaqMan probes has further advantages: (i) low background fluorescence due to the short distances between the reporters and quenchers increases the signal-to-noise ratio of the assay (31), and (ii) the small MGB molecules bind into the minor groove of double-stranded DNA, thus stabilizing the duplex, which results in higher melting temperatures and allows the application of probes that are shorter and more specific (13- to 18-mers) than standard probes (15- to 40-mers). As the target sequence of the assay, the F0 cleavage site, shows a high degree of variability on the nucleotide level, this is a great advantage. On the other hand, short MGB probes are more sensitive to base mismatches, since nonstandard base pairing leads to a more radical decline in melting-temperature values than in longer probes (Applied Biosystems product manual). In order to overcome this problem, degenerate primers and probes were applied for the detection of lentogenic and velogenic/mesogenic NDVs.

The specificity of the pathotyping assay was demonstrated by testing 39 NDV strains collected from various host species, time periods, and geographic regions. The panel of NDV isolates that we used in these experiments represented every genotype (I to VIII) and provided genetic variability at the nucleotide level that could not be achieved with a simple collection of clinical samples. The phylogenetic diversity of the samples was proven by the comparative nucleotide sequence analysis of the 325-bp-long fragment from the fusion protein genes (Fig. 1). In the specificity assay, 9 lentogenic and 30 velogenic/mesogenic strains were pathotyped successfully, every previously characterized NDV was detected by the corresponding primer-probe set, and no false-positive test result was obtained with the isolates belonging to the other pathotype. Similarly, no cross-reaction was observed with the 24 heterologous avian pathogens. In the corresponding RRT-PCR assays, the CT values varied between 14.7 to 34.0 and 12.6 to 30.8 for lentogenic and velogenic/mesogenic strains, respectively. Since the amount of template RNA was adjusted to within a rather narrow range, the observed variation in CT values can be explained by the differences in the efficiencies of RRT-PCRs for various NDVs. The exact molecular background of these alterations in PCR efficiency (e.g., the roles of secondary structures and noncomplementary base pairs in the primer/probe annealing sites) could be determined in further experiments focusing on this topic. In the specificity tests, 1- and, on one occasion (KR-5/99), 2-bp mismatches between the TaqMan MGB probe and target sequences were tolerated by the real-time PCR and resulted in a positive fluorescent signal. The application of degenerate probes (equivalent to 2 and 12 identical labeled oligonucleotides) and the observed tolerance of the PCR for noncomplementary bases greatly reduced the likelihood that any NDV remained undetectable or untypeable in the tests (in our experiments, BG-13/69 was the only isolate that resulted in very low relative fluorescence values). In such problematic cases, the assay could be adapted to the emerging new strains that might contain nonstandard F0 cleavage site motifs by introducing novel probes (4). Despite the convincing specificity results, caution is required in interpretation of the RRT-PCR data. The assay described here may not allow us to pathotype all NDV isolates, since the high degree of diversity in the nucleotide sequence of the F0 cleavage site may lead to false-negative test results when viruses with “nonstandard” motifs are examined (30). If necessary in future clinical applications, this uncertainty factor can be excluded by supplementing the assay with a PCR targeting a more conserved region of the genome (e.g., the matrix protein gene) (43), as was proposed by Kim et al. (30). It also has to be kept in mind that the method presented here was designed to differentiate between virulent and avirulent NDVs. This fact might be particularly important in some cases; for instance, mesogenic strains, such as H, Mukteswar, Komarov, and Roakin, are still being used for secondary vaccination in some countries, but these strains are detected by the PCR specific for velogens/mesogens (Table 3), in agreement with the definition of virus virulence by the OIE (6). In such situations, the vaccination protocol should be taken into consideration when RRT-PCR data are evaluated.

FIG. 1.

FIG. 1.

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Phylogenetic analysis of nucleotide sequences from NDV strains based on a 325-bp-long fragment amplified from the fusion protein genes, which encompasses the annealing sites of oligonucleotide primers and TaqMan MGB probes described in the RRT-PCR pathotyping assay and the coding region for the F0 proteolytic cleavage site. The 325-bp PCR fragment corresponds to nucleotide positions 4657 to 4981 in the NDV B1 complete genome (GenBank accession number AF309418). The phylogenetic tree was constructed by the neighbor-joining method (39) from the previously aligned sequences using the software package Treecon (42). Virulence properties and NDV genotypes (8, 16) are indicated after the name of the strain and on the right, respectively. len, lentogenic; mes, mesogenic; vel, velogenic.

In the time course experiment, when day-old chicks were experimentally infected by the intraocular route, the TaqMan MGB PCR was able to detect the vaccine strain PHY-LMV42 in significantly more specimens (14/21) than the traditional PCR (4/21) using the primer pair A and B (29). The analysis of RRT-PCR data allowed us to follow the spreading of the vaccine virus from the conjunctiva (2 days p.v.) to the respiratory/digestive systems (lung, trachea, and oral swab samples at 5 days p.v.) and later the gradual decrease of the viral load in the organ samples (9 days p.v.). At 9 days p.v., the vaccine virus was no longer circulating systemically in the inoculated chicks, but the strong fluorescent signal detected in the cloacal swab indicated extensive shedding in feces at this stage. The results obtained with the swab samples are in agreement with the data from a previously described real-time PCR assay targeting the fusion gene (43). In that experiment, oral and cloacal swab samples were tested following inoculation with a virulent NDV isolate, and 2 days after infection, a fluorescent signal was observed only in the oral swab samples, while at 4 days after infection, both samples were found positive. In the case of stool samples, when animals were experimentally infected with strains VG/GA and Hitchner B1, 100% correlation was observed between the results obtained with the pathotyping RRT-PCR and the gel-based PCR. The results suggest that the assay is suitable for clinical applications and that it can be a potential tool for the quantitative and qualitative measurements of viral load (both virulent and avirulent), either separately or simultaneously, e.g., in challenge experiments.

The detection limit of the assays was determined by using two different templates: plasmids carrying the F gene sequences from a lentogenic/velogenic isolate and cDNA prepared from allantoic fluid containing lentogenic/mesogenic NDV strains. According to the test results, the TaqMan MGB PCR was found to be as sensitive as (or more sensitive than) the previously described universal NDV-detecting/pathotyping PCR methods (21, 23, 29, 37, 43).

The novel RRT-PCR described in this article uses the TaqMan MGB platform for the pathotype prediction of NDV. The pathotyping assay is rapid and simple, and it involves only two reactions that can be performed simultaneously, one specific for virulent NDVs and the other for strains of low virulence. The reliability of the method is based on the careful design of PCR primers/probes, where the high degree of variability in the nucleotide sequence of the fusion gene was taken into consideration. In order to avoid false-negative test results, degenerate primer/probe sets were used in the assay to distinguish between lentogenic and velogenic/mesogenic strains in accordance with the OIE definition of virus virulence. The degenerate TaqMan MGB probes that were mainly responsible for the specificity of the reactions corresponded to several individual probes: FT_NDV_Lprobe2 (specific for lentogens; one degenerate position) and FT_NDV_Vprobe1 and -2 (specific for velogens/mesogens; three and two degenerate positions) were equivalent to 2 and 12 (8 plus 4) labeled oligonucleotides, respectively. In the PCR specific for lentogenic strains, the two individual probes targeted genotype I and II isolates, respectively, like the classic TaqMan probes 3A and 12A described by Aldous et al. (4). For velogens/mesogens, such probe specification could not be determined due to the more complex sequence variations in the F0 cleavage site region. By using degenerate primers and probes, the method has the potential to pathotype a broad range of NDVs from all known genotypes, including isolates with “nonstandard” F0 cleavage sites. Application of the lentogen-specific RRT-PCR lowered the risk that a virulent strain might escape the pathogenicity test, which can be further reduced by the introduction of a universal NDV-detecting PCR. The method was thoroughly tested with samples originating from different time periods, geographical locations, and avian hosts. The results indicate that the new assay is a useful alternative to the existing diagnostic protocols.

Acknowledgments

We thank B. Lomniczi (VMRI, Budapest, Hungary) for providing APMV isolates and plasmid constructs (pCR4-F and pKR5-FHN) for the PCR sensitivity tests and Z. Pénzes (Ceva-Phylaxia, Budapest, Hungary) for kindly supplying NDV vaccine strains. The contributions of J. Tanyi, S. Kecskeméti, and T. Bistyák, who collected virus and bacterial field isolates (heterologous pathogens) from clinical samples in the Department of Microbiology (Central Agricultural Office, Debrecen, Hungary), are also acknowledged. We are grateful to S. Egedi (Biomi, Gödöllő, Hungary) for support in oligonucleotide synthesis and DNA sequencing and to Donald King (Institute for Animal Health, Pirbright Laboratory, United Kingdom) for critical reading of the manuscript.

This work was supported by a grant from the 6th Framework Programme of the European Union (Lab-on-Site Project, contract no. SSPE-CT-2004-513645) and by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas, project no. 2006-2169), as well as by the partners of the scientific consortium of this EC project (http://www.labonsite.com).

Footnotes

▿

Published ahead of print on 13 May 2009.

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