- Email: [email protected]
The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection
Diagnostic Microbiology and Infectious Disease xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection Thomas J Gniadek a,1, Michael T. Forman a, Isabella Martin a,2, Ravit Arav-Boger b, Alexandra Valsamakis a,⁎ a b
Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD Pediatric Infectious Disease, Johns Hopkins School of Medicine, Baltimore, MD
a r t i c l e
i n f o
Article history: Received 26 January 2017 Received in revised form 19 May 2017 Accepted 26 May 2017 Available online xxxx
a b s t r a c t A patient developed disseminated adenovirus infection following bone marrow transplant. TaqMan real-time PCR showed reduced maximum fluorescence in the amplification curve from all plasma samples. Sequencing revealed three single nucleotide mismatches between the TaqMan probe and probe binding region. Real-time PCR with probe matching the isolate sequence showed normal amplification and a higher copy number result. © 2017 Elsevier Inc. All rights reserved.
Keywords: Adenovirus Aberrant real-time PCR Reduced fluorescence
A 13-year-old girl received a haploidentical allogeneic bone marrow transplant from her father (Day 0) to treat metastatic alveolar rhabdomyosarcoma. Induction included fludarabine, cyclophosphamide, and 200 cGy total body irradiation. Subsequently, she developed fever, Candida krusei fungemia (Days 10–17) treated with anti-fungal medication plus granulocyte transfusions, and BK virus-associated hemorrhagic cystitis. Engraftment was recognized on Day 19. By Day 21, she developed respiratory failure. Intravenous cidofovir (1 mg/kg per week) was initiated for BK virus treatment, but it was ineffective. She deteriorated further, with fever, diarrhea, and worsening respiratory failure requiring extracorporeal membrane oxygenation (Days 32–36). Although nasopharyngeal swab testing by multiplex PCR (Luminex, Austin, TX) had been negative for adenovirus prior to transplant and on Day 11, adenovirus infection was considered. Therefore, adenovirus DNA was measured in plasma using a real-time PCR laboratory developed test (LDT) targeting a conserved 104 bp region of the adenovirus hexon gene and utilizing a TaqMan hydrolysis probe (Heim et al., 2003). No adenovirus DNA was detected on Days 6, 29, or 32. Adenovirus DNA was detected on Day 36, below the limit of quantification. Subsequently, adenovirus DNA levels rose despite cidofovir treatment (Table 1). Cidofovir was discontinued on Day 55. An experimental antiviral medication was initiated on Day 60. Both viral load and patient
⁎ Corresponding author. Tel.: +1-410-955-5077. E-mail address: [email protected] (A. Valsamakis). Present Address: Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN. 2 Present Address: Department of Pathology and Laboratory Medicine, DartmouthHitchock Medical Center, Lebanon, NH. 1
Table 1 Quantification of adenovirus plasma viral load using TaqMan real-time PCR. Post-transplant day
6 29 32 36 41 44 48 52 55 60 63 68 70 74 76 81 83
Plasma adenovirus DNA (copies/ml), Quantified by real-time PCR Mismatched probea
Patient-specific probeb
Negative Negative Negative Positived Positived Negative 5220 (3.72) 1810 (3.26) 11,900 (4.08)
QNSe 543,966 (5.74) 878,178 (5.94)
505 (2.70)
114,681 (5.06)
29,600 (4.47)
163,584 (5.21)
4590 (3.66)
36,144 (4.56)
1300 (3.11)
4834 (3.68)
Reference labc
2,000,000 (6.30) 103,000 (5.01) 1,100,000 (6.04) 124,000 (5.09) 7300 (3.86)
a Assay based on published primers and probes (Heim et al., 2003). Calibrator was a plasmid sub-clone of an amplicon synthesized from real-time PCR of adenovirus serotype 2 strain. Arithmetic and log10 copy/mL data (parentheses) are shown. b Assay consisted of original, published real-time PCR primers and patient-specific probe. Calibrator was a plasmid sub-clone of an amplicon synthesized from real-time PCR of HADV5 strain. Arithmetic and log10 copy/mL data (parentheses) are shown. c External reference laboratory (Viracor Eurofins, Lee's Summit, MO) utilized a multitarget real-time PCR test to quantify adenovirus from plasma. Arithmetic and log10 copy/ mL data (parentheses) are shown. d Below the limit of quantitation. e Quantity of plasma not sufficient for testing.
http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013 0732-8893/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013
2
T.J. Gniadek et al. / Diagnostic Microbiology and Infectious Disease xxx (2017) xxx–xxx
10
A
Day-48 Day-52 Day-55
1
Day-63 (1:2) Day-70
Delta Rn
0.1
Day-76 Day-83
0.01 0.001 0.0001 0.00001 1
6
11
16
21
26
31
36
41
Cycle
B
10
100nM Mismatched Probe 250 nM Mismatched Probe
1
100nM Matched Probe
Delta Rn
250nM Matched Probe 0.1
0.01
0.001
0.0001 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Cycle
Fig. 1. TaqMan real-time PCR amplification curves from the patient's plasma. Panel A: HADV-5 plasmid controls (ten-fold dilutions, from 1 × 101 to 1 × 107 copies/reaction, gray dotted) compared to patient plasma (Days 48–83) demonstrated diminished maximum fluorescence with the original clinical TaqMan PCR probe (solid line). Panel B: Real-time PCR of the patient's HADV template (1 × 103 copies per reaction in a TA cloning vector) using the published, mismatched probe (100 nM) produced increased cycle-threshold and diminished maximum fluorescence (blue, dotted line) that corrected using patient-specific TaqMan PCR probe (100 nM; red, dotted line). Increasing the concentration of mismatched probe to 250 nM (blue, solid line) increased the maximum fluorescence and slightly decreased the cycle-threshold. There was no significant change in maximum fluorescence or cycle threshold after increasing a patient-specific probe concentration from 100 nM (red, dotted line) to 250 nM (red, solid line).
symptoms decreased in the subsequent weeks. Sanger sequencing of a variable region of the hexon gene and BLAST to Gen-Bank showed adenovirus serotype 5 (HADV-5) (Lu and Erdman, 2006). Amplification plots demonstrated aberrant curves with diminished maximum fluorescence for all of the patient's positive samples compared to positive control adenovirus (serotype type 2, HADV-2) when diagnostic testing was performed (data not shown) and compared to a plasmid sub-clone of commercially obtained HADV-5 (Fig. 1A). The curve abnormality was reproducible across different reagent lots (data not shown). In addition to aberrant curves with decreased maximum florescence, the LDT probe also resulted in amplification curves with a right shift (Fig. 1A and B), potentially resulting in falsely low copy number results. The patient's adenovirus real-time PCR LDT amplicon was too small for direct sequencing by capillary electrophoresis. Therefore, the amplified PCR product was sub-cloned into a plasmid vector (TA-cloning vector; Invitrogen, Carlsbad, CA). The amplification curve abnormality was reproducible using this sub-clone (Fig. 1B).
M13 primers external to the amplicon were used for sequencing with the Sanger method. Three single nucleotide mismatches in the probe binding region were identified in the patient's adenovirus compared to the published TaqMan probe sequence: G N A, position 18,913; G N C, position 18,919; and T N G, position 18,922 (Fig. 2). Isolates with two of these mismatches were reported in the publication describing this real-time PCR method (Heim et al., 2003). The third single nucleotide variant (G N A, position 18,913) has been found in other human adenovirus isolates (GenBank EU867462.1) (Biere and Schweiger, 2010). To investigate the role of these sequence polymorphisms on the efficiency of real-time PCR, two TaqMan 6FAM- and TAMRA- labeled probes were obtained commercially by custom synthesis (Thermo Fisher Scientific Inc., Waltham, MA). One matched the hexon sequence of a commercially-obtained HADV-5 (strain VR-5; ATCC, Manassas, VA) and differed from the patient's adenovirus hexon sequence at one position (G N A, position 18,913). The second probe matched the patient's adenovirus hexon sequence.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013
T.J. Gniadek et al. / Diagnostic Microbiology and Infectious Disease xxx (2017) xxx–xxx
3
Fig. 2. TaqMan probe and viral sequences. Panel A: Alignment of the original TaqMan probe with the patient's viral isolate and the sequence from the HADV-5 isolate obtained from ATCC. The three mismatches between the patient's isolate and the original probe are shown in red. Two of these mismatches (open arrows) were shared with the ATCC HADV-5, while one was not (solid arrow). Panel B: The sequence of the patient's viral isolate was compared to GenBank entries by BLAST. Using the patient's isolate, 100% matches were found for entries labeled Mastadenovirus C as well as human adenovirus type 5 and type 6. For these entries, the number of 100% matches found for the original probe and HADV-5 viral isolate sequences are shown.
The patient-specific TaqMan probe improved real-time PCR efficiency, as demonstrated by left-shifted curves of higher amplitude compared to mismatched probe (Fig. 1B); HADV-5 probe had a similar effect (data not shown). Additionally, the effect of probe mismatch could be partially overcome by increasing the mismatched probe concentration from 100 nM to 250 nM per reaction (Fig. 1B). In addition to an aberrant amplification curve, the mismatch between the patient's adenovirus sequence and LDT real-time PCR probe caused an under-estimation of the plasma adenovirus DNA concentration. Specifically, calculated plasma adenovirus DNA concentration was 4–300-fold higher upon re-testing with real-time PCR using a patient-specific probe (Table 1). The latter values were in agreement with those obtained with a multi-target real-time PCR performed by an external reference laboratory (Table 1). TaqMan real-time PCR fluorescence relies on efficient primer-based PCR amplification and probe hydrolysis. Factors that impair amplification and can cause diminished maximum fluorescence include reagent failure, template degradation, low template concentration, interfering substances, or sequence variations in the primer binding regions (Freeman et al., 1999; TaqMan Gene Expression Assays Protocol, 2010). Target sequence polymorphisms within the probe-binding region can lead to false negative results (Binnicker et al., 2013). Single nucleotide mismatches between the target and the probe can also cause an amplification curve with reduced maximum fluorescence, as reported with both traditional TaqMan probes and minor groove binding (MGB) probes (Whiley and Sloots, 2005; Yao et al., 2006). Importantly, abnormal amplification curves can lead to erroneous quantification and decrease the sensitivity of real-time RT-PCR for viral detection (Klungthong et al., 2010; Suss et al., 2009). Rectifying an abnormal amplification curve by sequencing a potential variant target followed by real-time PCR with a custom probe matching the target is impractical for most laboratories. Our data demonstrate that a simpler alternative may be to increase the probe concentration in real-time PCR reactions, which improved real-time PCR curve characteristics in the presence of a slight sequence mismatch. Multi-target assays may also be useful in circumventing real-time PCR abnormalities and quantification errors due to sequence variants. The efficacy of this approach has been demonstrated in commercial assays designed to overcome human immunodeficiency virus type 1 and hepatitis C virus sequence variants (Paryan et al., 2012) as well as in reports of tests designed to overcome targets with known sequence
variability, such as BK virus (Glover et al., 2016). In our case, values obtained by a reference laboratory that employed a multi-target realtime PCR assay agreed with those obtained by real-time PCR with the patient-specific probe. In summary, probe-target mismatch can result in diverse real-time PCR anomalies. Our case demonstrated reduced maximal fluorescence and increased cycle-threshold that could be overcome with custom probe matched to the patient's isolate or partially overcome with high concentrations of the mismatched probe. Furthermore, potential probe-target sequence mismatches should be considered when determining the concentration of probe to use in a clinical assay. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements The authors are grateful for the comments on the manuscript provided by Parvez Lokhandwala. References Biere B, Schweiger B. Human adenoviruses in respiratory infections: sequencing of the hexon hypervariable region reveals high sequence variability. J Clin Virol 2010;47: 366–71. Binnicker MJ, Baddour LM, Grys TE, Espy MJ, Hata DJ, Wotton JT, et al. Identification of an influenza a H1N1/2009 virus with mutations in the matrix gene causing a negative result by a commercial molecular assay. J Clin Microbiol 2013;51:2006–7. Freeman WM, Walker SJ, Vrana KE. Quantitative RT-PCR: pitfalls and potential. Biotechniques 1999;26:112–22. [124–115]. Glover WA, Atienza EE, Nesbitt S, Kim WJ, Castor J, Cook L, et al. Evaluation and utilization of preassembled frozen commercial fast real-time qPCR master mixes for detection of cytomegalovirus and BK virus. J Med Virol 2016;88:115–9. Heim A, Ebnet C, Harste G, Pring-Akerblom P. Rapid and quantitative detection of human adenovirus DNA by real-time PCR. J Med Virol 2003;70:228–39. Klungthong C, Chinnawirotpisan P, Hussem K, Phonpakobsin T, Manasatienkij W, Ajariyakhajorn C, et al. The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza a/H1N1/2009 virus detection by real-time RTPCR. J Clin Virol 2010;48:91–5. Lu X, Erdman DD. Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene. Arch Virol 2006;151:1587–602. Paryan M, Mohammadi-Yeganeh S, Mirab Samiee S, Rezvan H. Design and development of a multiplex real-time PCR assay for detection of HIV-1 and HCV using molecular beacons. Indian J Microbiol 2012;52:456–63.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013
4
T.J. Gniadek et al. / Diagnostic Microbiology and Infectious Disease xxx (2017) xxx–xxx
Suss B, Flekna G, Wagner M, Hein I. Studying the effect of single mismatches in primer and probe binding regions on amplification curves and quantification in real-time PCR. J Microbiol Methods 2009;76:316–9. TaqMan Gene Expression Assays Protocol. Applied Biopsystems by Life Technologies Corporation; 2010.
Whiley DM, Sloots TP. Sequence variation in primer targets affects the accuracy of viral quantitative PCR. J Clin Virol 2005;34:104–7. Yao Y, Nellaker C, Karlsson H. Evaluation of minor groove binding probe and Taqman probe PCR assays: influence of mismatches and template complexity on quantification. Mol Cell Probes 2006;20:311–6.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection Thomas J Gniadek a,1, Michael T. Forman a, Isabella Martin a,2, Ravit Arav-Boger b, Alexandra Valsamakis a,⁎ a b
Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD Pediatric Infectious Disease, Johns Hopkins School of Medicine, Baltimore, MD
a r t i c l e
i n f o
Article history: Received 26 January 2017 Received in revised form 19 May 2017 Accepted 26 May 2017 Available online xxxx
a b s t r a c t A patient developed disseminated adenovirus infection following bone marrow transplant. TaqMan real-time PCR showed reduced maximum fluorescence in the amplification curve from all plasma samples. Sequencing revealed three single nucleotide mismatches between the TaqMan probe and probe binding region. Real-time PCR with probe matching the isolate sequence showed normal amplification and a higher copy number result. © 2017 Elsevier Inc. All rights reserved.
Keywords: Adenovirus Aberrant real-time PCR Reduced fluorescence
A 13-year-old girl received a haploidentical allogeneic bone marrow transplant from her father (Day 0) to treat metastatic alveolar rhabdomyosarcoma. Induction included fludarabine, cyclophosphamide, and 200 cGy total body irradiation. Subsequently, she developed fever, Candida krusei fungemia (Days 10–17) treated with anti-fungal medication plus granulocyte transfusions, and BK virus-associated hemorrhagic cystitis. Engraftment was recognized on Day 19. By Day 21, she developed respiratory failure. Intravenous cidofovir (1 mg/kg per week) was initiated for BK virus treatment, but it was ineffective. She deteriorated further, with fever, diarrhea, and worsening respiratory failure requiring extracorporeal membrane oxygenation (Days 32–36). Although nasopharyngeal swab testing by multiplex PCR (Luminex, Austin, TX) had been negative for adenovirus prior to transplant and on Day 11, adenovirus infection was considered. Therefore, adenovirus DNA was measured in plasma using a real-time PCR laboratory developed test (LDT) targeting a conserved 104 bp region of the adenovirus hexon gene and utilizing a TaqMan hydrolysis probe (Heim et al., 2003). No adenovirus DNA was detected on Days 6, 29, or 32. Adenovirus DNA was detected on Day 36, below the limit of quantification. Subsequently, adenovirus DNA levels rose despite cidofovir treatment (Table 1). Cidofovir was discontinued on Day 55. An experimental antiviral medication was initiated on Day 60. Both viral load and patient
⁎ Corresponding author. Tel.: +1-410-955-5077. E-mail address: [email protected] (A. Valsamakis). Present Address: Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN. 2 Present Address: Department of Pathology and Laboratory Medicine, DartmouthHitchock Medical Center, Lebanon, NH. 1
Table 1 Quantification of adenovirus plasma viral load using TaqMan real-time PCR. Post-transplant day
6 29 32 36 41 44 48 52 55 60 63 68 70 74 76 81 83
Plasma adenovirus DNA (copies/ml), Quantified by real-time PCR Mismatched probea
Patient-specific probeb
Negative Negative Negative Positived Positived Negative 5220 (3.72) 1810 (3.26) 11,900 (4.08)
QNSe 543,966 (5.74) 878,178 (5.94)
505 (2.70)
114,681 (5.06)
29,600 (4.47)
163,584 (5.21)
4590 (3.66)
36,144 (4.56)
1300 (3.11)
4834 (3.68)
Reference labc
2,000,000 (6.30) 103,000 (5.01) 1,100,000 (6.04) 124,000 (5.09) 7300 (3.86)
a Assay based on published primers and probes (Heim et al., 2003). Calibrator was a plasmid sub-clone of an amplicon synthesized from real-time PCR of adenovirus serotype 2 strain. Arithmetic and log10 copy/mL data (parentheses) are shown. b Assay consisted of original, published real-time PCR primers and patient-specific probe. Calibrator was a plasmid sub-clone of an amplicon synthesized from real-time PCR of HADV5 strain. Arithmetic and log10 copy/mL data (parentheses) are shown. c External reference laboratory (Viracor Eurofins, Lee's Summit, MO) utilized a multitarget real-time PCR test to quantify adenovirus from plasma. Arithmetic and log10 copy/ mL data (parentheses) are shown. d Below the limit of quantitation. e Quantity of plasma not sufficient for testing.
http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013 0732-8893/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013
2
T.J. Gniadek et al. / Diagnostic Microbiology and Infectious Disease xxx (2017) xxx–xxx
10
A
Day-48 Day-52 Day-55
1
Day-63 (1:2) Day-70
Delta Rn
0.1
Day-76 Day-83
0.01 0.001 0.0001 0.00001 1
6
11
16
21
26
31
36
41
Cycle
B
10
100nM Mismatched Probe 250 nM Mismatched Probe
1
100nM Matched Probe
Delta Rn
250nM Matched Probe 0.1
0.01
0.001
0.0001 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Cycle
Fig. 1. TaqMan real-time PCR amplification curves from the patient's plasma. Panel A: HADV-5 plasmid controls (ten-fold dilutions, from 1 × 101 to 1 × 107 copies/reaction, gray dotted) compared to patient plasma (Days 48–83) demonstrated diminished maximum fluorescence with the original clinical TaqMan PCR probe (solid line). Panel B: Real-time PCR of the patient's HADV template (1 × 103 copies per reaction in a TA cloning vector) using the published, mismatched probe (100 nM) produced increased cycle-threshold and diminished maximum fluorescence (blue, dotted line) that corrected using patient-specific TaqMan PCR probe (100 nM; red, dotted line). Increasing the concentration of mismatched probe to 250 nM (blue, solid line) increased the maximum fluorescence and slightly decreased the cycle-threshold. There was no significant change in maximum fluorescence or cycle threshold after increasing a patient-specific probe concentration from 100 nM (red, dotted line) to 250 nM (red, solid line).
symptoms decreased in the subsequent weeks. Sanger sequencing of a variable region of the hexon gene and BLAST to Gen-Bank showed adenovirus serotype 5 (HADV-5) (Lu and Erdman, 2006). Amplification plots demonstrated aberrant curves with diminished maximum fluorescence for all of the patient's positive samples compared to positive control adenovirus (serotype type 2, HADV-2) when diagnostic testing was performed (data not shown) and compared to a plasmid sub-clone of commercially obtained HADV-5 (Fig. 1A). The curve abnormality was reproducible across different reagent lots (data not shown). In addition to aberrant curves with decreased maximum florescence, the LDT probe also resulted in amplification curves with a right shift (Fig. 1A and B), potentially resulting in falsely low copy number results. The patient's adenovirus real-time PCR LDT amplicon was too small for direct sequencing by capillary electrophoresis. Therefore, the amplified PCR product was sub-cloned into a plasmid vector (TA-cloning vector; Invitrogen, Carlsbad, CA). The amplification curve abnormality was reproducible using this sub-clone (Fig. 1B).
M13 primers external to the amplicon were used for sequencing with the Sanger method. Three single nucleotide mismatches in the probe binding region were identified in the patient's adenovirus compared to the published TaqMan probe sequence: G N A, position 18,913; G N C, position 18,919; and T N G, position 18,922 (Fig. 2). Isolates with two of these mismatches were reported in the publication describing this real-time PCR method (Heim et al., 2003). The third single nucleotide variant (G N A, position 18,913) has been found in other human adenovirus isolates (GenBank EU867462.1) (Biere and Schweiger, 2010). To investigate the role of these sequence polymorphisms on the efficiency of real-time PCR, two TaqMan 6FAM- and TAMRA- labeled probes were obtained commercially by custom synthesis (Thermo Fisher Scientific Inc., Waltham, MA). One matched the hexon sequence of a commercially-obtained HADV-5 (strain VR-5; ATCC, Manassas, VA) and differed from the patient's adenovirus hexon sequence at one position (G N A, position 18,913). The second probe matched the patient's adenovirus hexon sequence.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013
T.J. Gniadek et al. / Diagnostic Microbiology and Infectious Disease xxx (2017) xxx–xxx
3
Fig. 2. TaqMan probe and viral sequences. Panel A: Alignment of the original TaqMan probe with the patient's viral isolate and the sequence from the HADV-5 isolate obtained from ATCC. The three mismatches between the patient's isolate and the original probe are shown in red. Two of these mismatches (open arrows) were shared with the ATCC HADV-5, while one was not (solid arrow). Panel B: The sequence of the patient's viral isolate was compared to GenBank entries by BLAST. Using the patient's isolate, 100% matches were found for entries labeled Mastadenovirus C as well as human adenovirus type 5 and type 6. For these entries, the number of 100% matches found for the original probe and HADV-5 viral isolate sequences are shown.
The patient-specific TaqMan probe improved real-time PCR efficiency, as demonstrated by left-shifted curves of higher amplitude compared to mismatched probe (Fig. 1B); HADV-5 probe had a similar effect (data not shown). Additionally, the effect of probe mismatch could be partially overcome by increasing the mismatched probe concentration from 100 nM to 250 nM per reaction (Fig. 1B). In addition to an aberrant amplification curve, the mismatch between the patient's adenovirus sequence and LDT real-time PCR probe caused an under-estimation of the plasma adenovirus DNA concentration. Specifically, calculated plasma adenovirus DNA concentration was 4–300-fold higher upon re-testing with real-time PCR using a patient-specific probe (Table 1). The latter values were in agreement with those obtained with a multi-target real-time PCR performed by an external reference laboratory (Table 1). TaqMan real-time PCR fluorescence relies on efficient primer-based PCR amplification and probe hydrolysis. Factors that impair amplification and can cause diminished maximum fluorescence include reagent failure, template degradation, low template concentration, interfering substances, or sequence variations in the primer binding regions (Freeman et al., 1999; TaqMan Gene Expression Assays Protocol, 2010). Target sequence polymorphisms within the probe-binding region can lead to false negative results (Binnicker et al., 2013). Single nucleotide mismatches between the target and the probe can also cause an amplification curve with reduced maximum fluorescence, as reported with both traditional TaqMan probes and minor groove binding (MGB) probes (Whiley and Sloots, 2005; Yao et al., 2006). Importantly, abnormal amplification curves can lead to erroneous quantification and decrease the sensitivity of real-time RT-PCR for viral detection (Klungthong et al., 2010; Suss et al., 2009). Rectifying an abnormal amplification curve by sequencing a potential variant target followed by real-time PCR with a custom probe matching the target is impractical for most laboratories. Our data demonstrate that a simpler alternative may be to increase the probe concentration in real-time PCR reactions, which improved real-time PCR curve characteristics in the presence of a slight sequence mismatch. Multi-target assays may also be useful in circumventing real-time PCR abnormalities and quantification errors due to sequence variants. The efficacy of this approach has been demonstrated in commercial assays designed to overcome human immunodeficiency virus type 1 and hepatitis C virus sequence variants (Paryan et al., 2012) as well as in reports of tests designed to overcome targets with known sequence
variability, such as BK virus (Glover et al., 2016). In our case, values obtained by a reference laboratory that employed a multi-target realtime PCR assay agreed with those obtained by real-time PCR with the patient-specific probe. In summary, probe-target mismatch can result in diverse real-time PCR anomalies. Our case demonstrated reduced maximal fluorescence and increased cycle-threshold that could be overcome with custom probe matched to the patient's isolate or partially overcome with high concentrations of the mismatched probe. Furthermore, potential probe-target sequence mismatches should be considered when determining the concentration of probe to use in a clinical assay. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements The authors are grateful for the comments on the manuscript provided by Parvez Lokhandwala. References Biere B, Schweiger B. Human adenoviruses in respiratory infections: sequencing of the hexon hypervariable region reveals high sequence variability. J Clin Virol 2010;47: 366–71. Binnicker MJ, Baddour LM, Grys TE, Espy MJ, Hata DJ, Wotton JT, et al. Identification of an influenza a H1N1/2009 virus with mutations in the matrix gene causing a negative result by a commercial molecular assay. J Clin Microbiol 2013;51:2006–7. Freeman WM, Walker SJ, Vrana KE. Quantitative RT-PCR: pitfalls and potential. Biotechniques 1999;26:112–22. [124–115]. Glover WA, Atienza EE, Nesbitt S, Kim WJ, Castor J, Cook L, et al. Evaluation and utilization of preassembled frozen commercial fast real-time qPCR master mixes for detection of cytomegalovirus and BK virus. J Med Virol 2016;88:115–9. Heim A, Ebnet C, Harste G, Pring-Akerblom P. Rapid and quantitative detection of human adenovirus DNA by real-time PCR. J Med Virol 2003;70:228–39. Klungthong C, Chinnawirotpisan P, Hussem K, Phonpakobsin T, Manasatienkij W, Ajariyakhajorn C, et al. The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza a/H1N1/2009 virus detection by real-time RTPCR. J Clin Virol 2010;48:91–5. Lu X, Erdman DD. Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene. Arch Virol 2006;151:1587–602. Paryan M, Mohammadi-Yeganeh S, Mirab Samiee S, Rezvan H. Design and development of a multiplex real-time PCR assay for detection of HIV-1 and HCV using molecular beacons. Indian J Microbiol 2012;52:456–63.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013
4
T.J. Gniadek et al. / Diagnostic Microbiology and Infectious Disease xxx (2017) xxx–xxx
Suss B, Flekna G, Wagner M, Hein I. Studying the effect of single mismatches in primer and probe binding regions on amplification curves and quantification in real-time PCR. J Microbiol Methods 2009;76:316–9. TaqMan Gene Expression Assays Protocol. Applied Biopsystems by Life Technologies Corporation; 2010.
Whiley DM, Sloots TP. Sequence variation in primer targets affects the accuracy of viral quantitative PCR. J Clin Virol 2005;34:104–7. Yao Y, Nellaker C, Karlsson H. Evaluation of minor groove binding probe and Taqman probe PCR assays: influence of mismatches and template complexity on quantification. Mol Cell Probes 2006;20:311–6.
Please cite this article as: Gniadek TJ, et al, The effect of a genetic variant on quantitative real-time PCR in a case of disseminated adenovirus infection, Diagn Microbiol Infect Dis (2017), http://dx.doi.org/10.1016/j.diagmicrobio.2017.05.013