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Journal of Veterinary Diagnostic Investigation Vol. 18 Issue 6, 519-528
Copyright © 2006 by the American Association of Veterinary Laboratory Diagnosticians
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Full Scientific Reports

Effect of probe-site mismatches on detection of virulent Newcastle disease viruses using a fusion-gene real-time reverse transcription polymerase chain reaction test

L. Mia Kim, Claudio L. Afonso and David L. Suarez1

Correspondence: 1Corresponding Author: David L Suarez, DVM, PhD, Southeast Poultry Research Laboratory, USDA-ARS, 934 College Station Rd, Athens, GA 30605


   Abstract
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 Sources and manufacturers
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
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Virulent forms of Newcastle disease virus (NDV) are a major concern for poultry producers around the world and the rapid diagnosis of an outbreak is crucial to any control program. A validated real-time reverse transcription-PCR test (fusion test) directed at the fusion-cleavage site of NDV was developed to differentiate virulent Newcastle disease virus strains from those of low virulence, however one virulent isolate, Dove/Italy/2736/2000, escaped detection during the initial evaluation of the test. The objectives of this study were to determine how this isolate differed from other detectable isolates, to identify other isolates that may fail detection, and to characterize the effect of specific probe-site mutations on the fusion test at a range of annealing temperatures. Using a virulent NDV isolate (Game fowl/US(CA)/2002) as a backbone that has 100% identity to the fusion-test probe, specific changes were made to the fusion-test probe-site to reflect the unique mismatches found in Dove/Italy/2736/2000 and other selected regions of the probe. Mutated clones with mismatches unique to Dove/Italy/2736/2000 at positions 6, 13, and 14 were not detected until annealing temperatures were lowered to 50°C. Those detected at 58°C contained 1–2 mismatches (position 1 and 6, 13 and 14, or 14 only) although increased cycle threshold values compared to the parent clone indicated decreased sensitivity. Data from this study predicts that the fusion test may fail to detect some viruses among lineage 4b and potential solutions to identify this subset of viruses include lowering the annealing temperature or modifying the probe.

Key Words: Fusion gene • Newcastle disease virus • pigeon paramyxovirus-1 • real-time RT-PCR • virus detection


   Introduction
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 Abstract
 Introduction
Materials and Methods
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Virulent forms of Newcastle disease virus (NDV) are a major concern for poultry producers around the world and the rapid diagnosis of an outbreak is crucial to any control program.3,7,10 The disease is reportable to the World Organization for Animal Health (Office International des Epizooties, OIE) if the intracerebral pathogenicity index is ≥0.7 on a 3-point scale, which includes both mesogenic and velogenic strains of virus. Outbreaks of NDV must also be rapidly identified and distinguished from other respiratory pathogens such as avian influenza virus. The development of real-time reverse transcription-polymerase chain reaction (RRT-PCR) for use in rapid diagnosis of human and animal diseases during the past 10 years12 provides the ability to obtain results from a field sample within 3–4 hours (depending on the virus) and has been invaluable for diagnostic laboratories in need of quick answers.

There are two US Department of Agriculture (USDA) validated RRT-PCR rapid diagnostic tests for NDV. A matrix-gene test (matrix test) targets conserved areas in the matrix gene and detects most avian paramyxovirus-1 (APMV-1) strains including pigeon paramyxoviruses (PPMV-1) regardless of pathotype and it is used as a primary screening test for NDV. If the matrix test is positive, then the sample is further tested with a fusion-gene test (fusion test), which is directed at the fusion-cleavage site of NDV and was developed to differentiate virulent (velogenic and mesogenic) Newcastle disease virus strains (vNDV) from those of low virulence (lentogenic and vaccine viruses).14,23 This test was used during the California 2002–2003 outbreak and demonstrated both efficiency and effectiveness.6 The fusion test has good sensitivity and specificity, identifying most but not all virulent viruses in the test panel used for validation. The one exception identified in the panel was a dove isolate, Dove/Italy/2736/2000 (DoveIT).20,23 DoveIT is phylogenetically related to PPMV-1 isolates represented in lineage 4b/VIb.2 Other pigeon isolates from lineage 4b/V1b have been successfully detected by the fusion test and further investigation was warranted to determine why this isolate escaped detection.15,23

The inability to diagnose any NDV isolate with RRT-PCR is an important area of investigation to further the understanding of how the test should be applied and provide guidance for clinicians and laboratory diagnosticians when dealing with unusual isolates. The specificity of molecular diagnostic tests is based on the nucleotide base-pairing of the primers and probe with sequence mismatches having the potential to produce false negative results. Ideally, molecular diagnostic tests are directed at highly conserved areas of the genome, but for tests that target a particular virulence motif such as the fusion test, this is the only area that can be targeted. When the sequence of the DoveIT isolate was compared to the sequences of the fusion-test primers and probes, several mismatches were identified in the primer sequences as well as 4 nucleotide mismatches at the fusion-test probe-site. The objectives of this study were to determine how this isolate differed from other detectable isolates, to identify other isolates that may fail to be detected, and to characterize the effect of specific mutations at the probe-site on the fusion test at a range of annealing temperatures. This information is critical in determining how the test should be used and to provide guidance on how the test can be improved.


   Materials and Methods
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 Abstract
 Introduction
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Results
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Isolates and Sequence Data
All viruses used in this study have been previously characterized.15,18 Sequence information used for alignments, nucleotide frequency, and entropy calculations were either from published data or from Genbank®. All available Genbank sequences for the genomic region encoding the 374-bp fusion gene were used (n = 970 duplicates excluded; accession numbers available upon request).

Preparation of Cdna, Clones, and in Vitro Transcribed Rna
The cDNA for the parent clone (Game fowl/US(CA)/2002, velogen, identical probe-site nucleotide sequence to the fusion-test probe) and control clone (Chicken/US/B1/48, lentogen) was prepared from extracted RNAa using primers targeting a 1,195-bp region spanning the matrix and fusion genes (forward primer M629F 5' TCG AGG NCT GTA CAA TCT TGC 3' position 3,884–3,903 on the full length NDV genome; reverse primer F581R 5' CTG CCA CTG CTA GTT GGA TAA TCC 3' position 5,054–5,078). A kit was used for RT-PCR amplificationb following the manufacturer's protocol. PCR products were clonedc and plasmids extracted.d Quantitation and purity estimations were performed with spectrometry and use of A260/A280 ratios. The plasmid inserts were sequenced to ensure accuracy. Subsequent testing was performed on serial 10-fold dilutions prepared from a stock concentration of 84 ng/µl (1.6 x 1010 copies/µl) for all clones. In vitro transcribed RNA was generated from the parent clone for testing with real-time RT-PCR using a kite per the manufacturer's protocol. A stock concentration of 125 pg/µl (2.0 x 108 copies/µl) was prepared.

Mutations to the Fusion-test Probe-site
The alignment at the fusion-test probe-site (Fig. 1) identified unique differences in the DoveIT probe-site when compared to other vNDV. Using the Game fowl/US(CA)/2002 as the backbone to ensure that any differences in detection by real-time PCR (R-PCR) would be due only to changes at the fusion-test probe-site, 5 permutations of the DoveIT probe-site and 2 additional mutations were generated for this study (Fig. 2). The probe-site mutations generated were based upon data from both the alignment of DoveIT (Fig. 1) and the entropy data for the virulent NDV isolates (Fig. 3a, n = 864). Mutations designated with (CA) indicate the Game fowl/US(CA)/2002 backbone and were as follows: "DoveIT(CA)" all 4 probe-site mismatch positions of DoveIT 1:T to A, 6:G to T, 13:A to G, and 14:C to A; "Dove–1(CA)" position 14:C to A; "Dove–2(CA)" positions 13–14:A to G and C to A; "Dove+2(CA)" positions 1,6:T to A and G to T; "Dove–3(CA)" positions 6,13–14:G to T, A to G and C to A; "5'(CA)" 3 nucleotides at positions 1–3 TGG to CAT; and "3'(CA)" 3 nucleotides at positions 22–24 CTT to TCA (Fig. 2). For negative controls, 2 additional clones were generated; one contained the lentogenic Chicken/US/B1/48 probe-site with the Game fowl/US(CA)/2002 backbone (B1(CA)), and the other clone contained the entire 1,195-bp segment from Chicken/US/B1/48 (B1 clone). Site-directed mutagenesis using custom-made mutagenic primers (sequence available upon request) were used to change the fusion-test probe-site of the parent clone (Game fowl/US(CA)/2002) with either a kitf as per the manufacturer's protocol or a manual method as follows: primer M629F with mutagenic 3' and mutagenic 5' with F581R were used to generate 2 separate products using standard PCR reactionsg, the products were purifiedh and combined in a third PCR reaction with the M629F and F581R primers. This resultant product was purified and cloned.c The fusion and matrix test primer sites were identical for all clones except for the B1 clone control. All clones and mutants were sequenced and aligned throughout their length (1,195 bp, matrix to fusion region) to verify successful cloning and mutation as well as absence of unwanted mutations (Fig. 2, 24-bp fusion-test probe-site only).


Figure 18060101
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  		Figure 1 Fusion-test probe-site alignment of 21 NDV isolates for comparison to Dove/Italy/2736/2000; positions 4,871–4,894 on the full length genome: 1–24 in figure. Arrow denotes fusion cleavage site.

 

Figure 18060102
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  		Figure 2 Fusion-test probe-site alignment for all 10 clones and mutations (designated "[CA]"). Data corresponding to the Dove/Italy/2736/2000 isolate is bolded. Results for the real-time PCR fusion test at the validated temperature of 58°C shown at right. Arrow denotes fusion cleavage site.

 

Figure 18060103
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  		Figure 3 a, Percent composition by nucleotide for the fusion-test probe-site of virulent Newcastle disease viruses and heterogeneity at each probe-site position calculated using the Shannon entropy algorithm (alternate y-axis) for positions 4,871–4,894 on the full length genome: 1–24 in figure, n = 864. b, Percent composition by nucleotide for the fusion-test probe-site of lentogenic Newcastle disease viruses and heterogeneity at each probe-site position calculated using the Shannon entropy algorithm (alternate y-axis) for positions 4,871–4,894 on the full length genome: 1–24 in figure, n = 106.

 
Nucleotide Sequencing, Alignment Analysis, and Entropy Data
All double-stranded nucleotide sequencing reactions were performed with fluorescent dideoxynucleotide terminators in an automated sequencer.i Nucleotide sequence editing and analysis were conducted with the LaserGene sequence analysis software package.j The alignment of the fusion probe-site (24 bp) was obtained by using ClustalW21 followed by manual editing with the BioEdit Sequence Alignment Editor for 970 previously described NDV isolates.9 The variability between the sequence of these NDV isolates and each position in the fusion-test probe-site was calculated by using the entropy algorithm available from the BioEdit software. This algorithm as defined by Shannon is H(l) = –{Sigma}f(b,l) ln[f(b,l)] where H(l) is the uncertainty (entropy) at position l, b represents a residue from the allowable choices for the sequence in question (A,C,G,T, – ), and f(b,l) is the frequency at which residue b is found at position l.16,17

Real-time Pcr (r-pcr) and Rrt-pcr
The R-PCR used in this study was adapted from the RRT-PCR protocols developed for the fusion (F+4839 5'-GGT GAG TCT ATC CGG ARG ATA CAA G-3', F-4939 5'-AGC TGT TGC AAC CCC AAG-3', probe F-4894 5'-[FAM ] AAG CGT TTC TGT CTC CTT CCT CCA [BHQ]-3') and matrix (M+4100 5'-AGT GAT GTG CTC GGA CCT TC-3', M-4220 5'-CCT GAG GAG AGG CAT TTG CTA-3', probe M+4169 5'-[FAM ] TTC TCT AGC AGT GGG ACA GCC TGC [BHQ]-3') test sets and contained identical concentrations of MgCl2 (3.75 mM), primers (fusion: 1.07 µM forward and 0.54 µM reverse, matrix: 0.4 µM forward and reverse), and probe (fusion: 0.18 µM, matrix: 0.24 µM) for 25 µl reactions in a standardized PCR master mix.g,14,23 All R-PCR and RRT-PCR reactions were performed using the SmartCycler®k and cycling conditions were identical to that of the previously reported fusion and matrix test protocols: the RT step was 30 min at 50°C (excluded for R-PCR) followed by 15 min at 95°C (2 min at 95°C for R-PCR); cycling conditions were 40 cycles of 10 s denaturation at 94°C, 30 s of annealing at 58°C for fusion and 56°C for matrix, and extension at 72°C for 10 s.14,23 The cycle threshold value (Ct) cut-off was set at 35 for all R-PCR and RRT-PCR. For R-PCR, the amplification curves obtained from the parent clone were comparable in slope and amplitude to those obtained with Game fowl/US(CA)/2002 RNA using RRT-PCR.23 All clones containing the Game fowl/US(CA)/2002 backbone were tested using the matrix R-PCR test at the validated temperature of 56°C to confirm uniformity among clones with identical primer and probe sites. Further testing was performed at a range of annealing temperatures (48–66°C) for the fusion test only.


   Results
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Significance of the Mismatches at the Doveit Fusion-test Probe-site
Sequence analysis of the DoveIT isolate identified 4 nucleotide differences at the probe-site when compared to 20 other isolates that were accurately detected by the fusion test (positive for velogens and mesogens and negative for lentogenic viruses), with mismatches corresponding to positions 1, 6, 13, and 14 (Fig. 1). Members of the PPMV-1 genotype that were known to be detected by the fusion test shared only 2 of the 4 nucleotide mismatches (positions 1 and 13) when compared to DoveIT.15,23 To further characterize the variability present by nucleotide position at the fusion probe-site (24 bp), nucleotide sequences from a total of 970 NDV isolates (864 virulent and 106 lentogenic) were analyzed for composition and heterogeneity at each position of the fusion-test probe-site (Fig. 3a, 3b). Heterogeneity expressed as entropy determines how accurately the sequence can be predicted at a specific site.16,17 Examination of this data revealed that sites of highest entropy or heterogeneity for virulent isolates were associated with positions 1, 13, and 16. In contrast, heterogeneity for lentogenic viruses was highest at positions 1, 7, 12, 19, and 22. Differences in nucleotide composition can clearly be seen between virulent and lentogenic strains at positions 7–8, 12–13, 17–19, and 23. These differences contribute to the ability to distinguish between virulent and lentogenic viruses using the fusion test.

The sites unique to DoveIT were also associated with increased entropy or heterogeneity, particularly positions 1 and 13 (Table 1; 1.18 and 0.65 respectively). Among the virulent isolates, the mismatches of DoveIT at positions 6 and 14 occur infrequently (3.2% and 3.8% respectively), and the mismatches at positions 1 and 13 represent approximately one-third (33.9% and 28.9% respectively) of the isolates evaluated. As mentioned previously, DoveIT is phylogenetically related to the PPMV-1 genotype, which are represented in the diverse lineage classified as 4b/VIb.2,22 Of the 970 isolates reviewed for this study, 38 isolates contained the DoveIT fusion-cleavage site motif "RRKKRF." The majority of these isolates originated in Europe with a small subset from Japan and 84% of these isolates were from pigeons.


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  		Table 1 Frequency and entropy (H[x]) of nucleotides for 864 virulent isolates at positions unique to Dove/Italy/2736/2000 (bolded numbers) for the 24-bp fusion-test probe-site.

 
To further characterize this small subset of PPMV-1 viruses, an alignment was produced from the nucleotide region corresponding to the fusion-test probe-site for these 38 isolates (Fig. 4). When compared to the fusion-test probe sequence, 86% of these isolates (32/37) contained identical mismatches to DoveIT. The other 8 isolates differed either at position 6, a highly variable site as defined previously, or position 16. In comparison to other virulent isolates reviewed in this study (n = 864), the 7 isolates that differed at position 6 (Table 1), 4 of 7 had G (represented in 93% of other virulent isolates) and 3 of 7 contained C, which was a rare occurrence among other virulent isolates (0.3%). The final isolate differed at position 16 (A), which occurred infrequently among other virulent isolates (Fig. 3a, 15.6% contain A at position 16).


Figure 18060104
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  		Figure 4 Fusion-test probe-site alignment representing 38 sequences comparing Dove/Italy/2736/2000 with 37 Genbank sequences that contain the fusion-cleavage site motif "RRKKRF" from lineage 4bii/VIb1 (Aldous, et al. 2003; Ujvari, et al. 2003). One reference sequence is presented for subsequent identical sequences. Arrow denotes fusion cleavage site. a Identical to 27 other pigeon isolates from 8 countries (UK, Ireland, France, Spain, Austria, Denmark, Japan, and South Africa). b Identical to 3 other pigeon isolates from Germany and Austria.c Identical to 1 other pigeon isolate from Japan.

 
This data together with the entropy data (Table 1) provided evidence that the fusion-test probe-site mismatches specific to DoveIT were frequent among a subset of PPMV-1 isolates and revealed increased variability at the 5' and 3' end of the probe-site thus warranting further investigation to determine the effect of probe-site mutations on detection with the fusion test.

Significance of Mismatches at the Primer Sites
An alignment at the fusion-test primer sites (sense and antisense) of 21 virulent and lentogenic viruses is shown in Fig. 5a, 5b. Twenty of the 21 viruses (95%) included in this alignment were accurately detected by the current fusion test (positive for velogens and mesogens and negative for lentogenic viruses) with the exception of DoveIT.23 The discrepancies identified in DoveIT were present in other virus sequences that were readily detected by the fusion test. Additionally, a product of the expected size was produced when DoveIT was amplified with the fusion-test primers by conventional RT-PCR (data not shown). These data provided evidence that the nucleotide differences identified in the primer regions were not responsible for failure of amplification and should not cause failure of detection with the fusion test.


Figure 18060105
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  		Figure 5 a, Fusion test forward (sense) primer-site alignment of 21 NDV isolates for comparison to Dove/Italy/2736/2000. b, Fusion test reverse (anti-sense) primer-site alignment of 21 NDV isolates for comparison to Dove/Italy/2736/2000 (in bold).

 
Comparison of R-pcr to Rrt-pcr
To determine whether testing with R-PCR using plasmid DNA was comparable to testing RNA with RRT-PCR, serial 10-fold dilutions of the parent clone DNA (starting with 840 pg/µl, 1.6 x 108 copies/µl) and of the in vitro transcribed RNA (ivtRNA; starting with 12.5 pg/µl, 2.0 x 107 copies/µl) were tested with fusion-test R-PCR and real-time RT-PCR, respectively (Fig. 6). The Ct values were tightly clustered with nearly equivalent slopes (m = –3.39 for plasmid DNA and m = –3.34 for ivtRNA, R2 = 0.99 for both). These data suggest that RNA and plasmid DNA have similar amplification efficiencies and results using plasmid DNA may be readily extrapolated to RRT-PCR. Additionally, RNA extracted from DoveIT allantoic fluid was tested using the fusion test at decreasing annealing temperatures and was not detected until annealing temperatures were decreased to ≤50°C.


Figure 18060106
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  		Figure 6 Comparison of Ct values for real-time PCR using the plasmid cDNA (from 840 pg/µl; 1.6 x 108 copies/µl) to real-time RT-PCR using in vitro transcribed RNA (from 12.5 pg/µl; 2.0 x 107 copies/µl) from the parent clone: Game fowl/US(CA)/02.

 
R-pcr Fusion-test Results
All 10 clones were tested in duplicate under a range of annealing temperatures from 66 to 48°C in 2° increments and the average of the Ct values was recorded (Table 2, data shown for 58–48°C). Mutated clones demonstrating comparable patterns of detection with decreasing annealing temperatures from 58 to 48°C and Ct value differences <1 were represented by a single clone: Dove+2(CA) and Dove–2(CA) had similar patterns and Ct values as those observed for Dove–1(CA); 5'(CA) data was representative of the 3'(CA) mutation; and data for B1(CA) was equivalent to that of the B1 clone. The specificity of the fusion test was ensured with the 2 negative control clones (B1[CA] and B1 clone), which tested negative at all dilutions and all annealing temperatures.


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  		Table 2 Real-time PCR fusion-test cycle threshold (Ct) values from representative mutated clones (bolded names) by decreasing annealing temperature from the validated temperature of 58°C. The Ct value cut-off was set at 35.

 
At the validated temperature of 58°C, the parent clone was detected down to 1.6 x 102 copies/µl (8.4 x 10–4 pg/µl) and 5 of the 7 mutated clones (Dove–1[CA], Dove+2[CA], Dove–2[CA], 5'[CA], and 3'[CA]) were detected down to 1.6 x 103 copies/µl (8.4 x 10–3 pg/µl) although increased Ct values (later detection) compared to the parent clone indicated decreased sensitivity (Table 2). The 3' clone appeared to be the most tolerated and averaged within 1 Ct value compared to the parent clone. The 3 variants of DoveIT detected at 58°C and the 5'(CA) mutation were detected an average of 2.2 Ct values later compared to the parent clone. The two remaining clones, Dove–3(CA) and DoveIT(CA) mutants both failed detection at 58°C. Therefore, mismatches at positions 6, 13, and 14 (Dove–3[CA]) were sufficient to prevent the successful binding of the probe.

For annealing temperatures from 58 to 48°C, 5 of the 7 mutated clones (Dove–1[CA], Dove+2[CA], Dove–2[CA], 5'[CA], and 3'[CA]) were detected from 58 to 52°C (Table 2). Dove–3(CA) and DoveIT(CA) mutants appeared to be the least tolerated and were not detected until the annealing temperature was lowered to 50°C. An average increase of 3.3 Ct values with increasing dilution within the same mutation was demonstrated by all mutated clones with the exception of DoveIT(CA), which averaged 4.5 Ct values between dilutions. Additionally, the Dove–3(CA) mutated clone was detected an average of 4.6 Ct values earlier than the DoveIT(CA) clone indicating a differential sensitivity between these mutations based on a single mismatch.

The effect of decreasing annealing temperature was consistent for all clones with greater variation in Ct values between dilutions found at temperatures above 58°C; results for the parent clone only are shown in Fig. 7. In general, earlier Ct values were noted for the same clone with decreasing annealing temperatures. The parent clone was detected down to 1.6 x 105 copies/µl (8.4 x 10–1 pg/µl) at all annealing temperatures (48–66°C). For temperatures above 58°C, 3 of the 5 DoveIT clones (Dove–1[CA], Dove+2[CA], Dove–2[CA]) were detected up to 60°C, the 5'(CA) clone was detected up to 62°C, and the 3'(CA) mutated clone was most easily tolerated and was detected down to 1.6 x 103 copies/µl (8.4 x 10–3 pg/µl) at 64°C (data not shown).


Figure 18060107
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  		Figure 7 Real-time PCR fusion test of the parent clone (Game fowl/US(CA)/02) at decreasing annealing temperatures by serial 10-fold dilutions (from 1.3 x 107 copies/reaction). The cycle threshold cut-off (Ct value) was set at 35. Line equation for annealing temperature of 58°C is shown.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Virulent NDV is one of the most important poultry diseases worldwide. Development of a single rapid test for the detection of vNDV has proven to be challenging because of the genetic diversity present in the genome.7,8,10,11,13,19 Although this nucleotide diversity provides the ability to differentiate virulent isolates from those that are not when using the fusion test, it may also be problematic when attempting to detect unusual viruses.1,4,5,23 For this reason the matrix test is the primary screening test, with the fusion test providing the confirmation for virulent viruses. The matrix test targets a more conserved region of the genome and identifies most NDV isolates including the subset of PPMV-1 isolates reviewed in the present study. If a poultry flock had clinical signs of vNDV and samples were NDV matrix-test positive and fusion-test negative, additional tests would need to be performed to rule out vNDV as the causative agent.

In the current study, a single isolate that consistently escaped detection by the validated fusion test was investigated. The DoveIT isolate was found to be phylogenetically related to PPMV-1 represented in lineage 4b/VIb.2,22 Some isolates from this lineage were known to be detected by the fusion test, but our analysis showed that pigeon paramyxoviruses that tested positive shared only 2 of the 4 nucleotide mismatches (at positions 1 and 13) when compared to DoveIT.15,23 Further examination of the isolates containing the fusion-cleavage motif "RRKKRF" placed them in a distinct subset: group 4bii, subgroup e.2 Eighty-six percent of the viruses in this subset had identical fusion-test probe-site mismatches when compared to DoveIT, and viruses from this subset are unlikely to be detected by the fusion test the way it is currently performed.

The use of R-PCR in this study provided an economical and efficient method of determining which mismatches in the DoveIT fusion-test probe-site led to failure of detection. Every effort was made to ensure that the conditions and reagent concentrations for the R-PCR were equivalent to that of the validated fusion test. As seen in the results, Ct values for the parent clone and its in vitro transcribed RNA were comparable within 1 log concentration for fusion-test R-PCR and RRT-PCR (Fig. 6). Further confirmation of the findings was performed by testing the DoveIT RNA on RRT-PCR at decreasing annealing temperatures.

An interesting finding from this study was the sequence variability between virulent and lentogenic strains (Figs. 3a, 3b). The fusion-test probe-site sequence comparison of 106 lentogenic viruses versus 864 virulent strains indicated that the nucleotide composition of lentogens differed considerably at positions 7–8, 12–13, 17–19, and 23. This information supports the specificity of the fusion-test probe, and when combined with the data from decreasing annealing temperatures, suggests that lentogenic viruses would not be recognized at temperatures as low as 48°C as shown with the B1 clone in this study.

Data from this study suggests that the annealing temperature of the fusion test could potentially be lowered without concern for false positive detection of lentogenic viruses. This approach may also be effective at detecting other unusual virulent isolates without decreasing the test specificity. However, unforeseen primer-probe interactions may be produced by lowering the annealing temperature requiring the use of alternative methods to solve the issue. One solution may be to design a degenerate probe that could identify the viruses in question or to have 2 different probes in a multiplex reaction. As seen in the results, the DoveIT(CA) mutant and the Dove–3(CA) mutant remained undetected until the annealing temperature was decreased to ≤50°C. Therefore, mismatches at position 6, 13, and 14 (Dove–3[CA]) were sufficient to prevent the successful binding of the probe, but adding the fourth mutation (as in DoveIT[CA]) increased the Ct values by >4 indicating the mismatch of a single nucleotide could influence the test sensitivity. Results for the mismatches at the 5' and 3' ends of the probe were comparable to the parent clone demonstrating that these mismatches were well tolerated for the fusion test. Therefore, efforts to introduce degeneracy in the design of future probes for the fusion test to increase detection of unusual isolates should be concentrated in the central region.

A degenerate probe with as little as a single nucleotide change may adequately identify members of the PPMV-1 subset in question. Among the virulent isolates reviewed in this study, the mismatches of DoveIT at positions 6 and 14 occurred infrequently (3.2% and 3.8% respectively), and the mismatches at positions 1 and 13 were readily detected by the fusion test. Therefore, position 6 would be the most plausible candidate for a degenerate site in the fusion-test probe.

Data from the current study predicts that the fusion test may fail to detect some viruses among lineage 4bii and potential solutions to identify this subset include lowering the annealing temperature or altering the fusion probe to include some degeneracy with position 6 being a likely candidate. This information is critical in determining how the test should be used and to provide guidance on how the test can be improved. Further investigation into the effect of sequence variation at both the probe and primer sites on the performance of rapid diagnostic tests such as RRT-PCR may assist in improving their sensitivity and specificity.


   Sources and manufacturers
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Abstract
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From the USDA, Agricultural Research Service, Southeast Poultry Research Laboratory, Athens GA. Back

a. Trizol LS; Invitrogen, Carlsbad, CA. Back

b. The Qiagen One-Step RT-PCR kit; Qiagen, Valencia, CA. Back

c. TOPO-TA; Invitrogen; Carlsbad, CA. Back

d. QIAprep® Spin Miniprep kit; Qiagen, Valencia, CA. Back

e. RiboMAXTM kit; Promega, Madison, WI. Back

f. Stratagene QuickChangeTM Site-Directed Mutagenesis; La Jolla, CA. Back

g. PCR MasterMix 2X; Promega, Madison, WI. Back

h. QIAquick® Gel extract kit; Qiagen, Valencia, CA. Back

i. ABI 3700 automated sequencer; Applied Biosystems Inc., Foster City, CA. Back

j. LaserGene, version 5.07; DNAStar, Inc., Madison, WI. Back

k. SmartCycler®; Cepheid, Inc., Sunnyvale, CA. Back


   References
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 Abstract
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 Materials and Methods
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 References
 

  1. Aldous E.W., Collins M.S., McGoldrick A., et al.: 2001, Rapid pathotyping of Newcastle disease virus (NDV) using fluorogenic probes in a PCR assay. Vet Microbiol 80:201–212.[Medline]
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