LETTER TO THE EDITOR
Frozen Master Mix-Based Modification of Commercial Reverse Transcriptase PCR for Detection of Influenza and Respiratory Syncytial Viruses Brooke Wenninger,a Erik Munson,a,b Maureen Napierala,a Kimber L. Munson,a Dorothy Bilbo,a Jeanne E. Hryciuka Wheaton Franciscan Laboratory, Milwaukee, Wisconsin, USAa; College of Health Sciences, University of Wisconsin—Milwaukee, Milwaukee, Wisconsin, USAb
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TABLE 1 Mean CT values obtained with replicates of prepared ProFlu⫹ positive-control material frozen for 2, 4, 8, or 13 weeks at ⫺70°Ca Mean CT value obtained after storage for: Control Influenza A virus Influenza B virus RSV A RSV B
0 wkb c
29.79 30.08c 28.88c 29.79c
2 wk
4 wk
8 wk
13 wk
29.14 28.03 27.71 29.47
28.83 27.82 27.54 29.40
28.88 27.89 27.25 29.17
29.20 28.15 27.90 29.64
a Temporal differences in master mix potency for each control were assessed by oneway analysis of variance. b Fresh master mix. c Fresh master mix P ⱕ 0.0001 versus all intervals of master mix freezing.
cacy (2, 3) and workflow (4) perspectives. Within the ProFlu⫹ assay, the catalyst for nucleic acid amplification is Moloney murine leukemia virus (MMLV) RT (5). The molecule itself is labile; i.e., nonionic detergents and glycerol are requirements for in vitro solubility (6). Package insert data emphasize that the master mix (components of which include separately packaged MMLV RT and an RNase inhibitor) must be prepared fresh for each RT-PCR run (1). However, the experiments presented demonstrate that prolonged frozen storage of prepared ProFlu⫹ control and master mix reagents at ⫺70°C yielded accurate results; in some instances, potency greater than that obtained with fresh master mix was observed. A paucity of literature exists with respect to extended stability of frozen MMLV RT; one report in the Chinese literature (7) suggests that a laboratory-developed RNA amplification assay exhibited appropriate stability when reagents were stored for up to 6 months at ⫺20°C. Furthermore, small-scale experiments not presented in this report reveal that frozen ProFlu⫹ master mix and controls possess sufficient potency following 12 to 15 months of storage at ⫺70°C. Pending in-house verification studies, these data provide laboratories an additional option for accurate and efficient detection of respiratory virus infection agents. Following introduction of the verified frozen master mix paradigm into routine laboratory practice, a reduction of average indeterminate-result rates from
Accepted manuscript posted online 28 January 2015 Citation Wenninger B, Munson E, Napierala M, Munson KL, Bilbo D, Hryciuk JE. 2015. Frozen master mix-based modification of commercial reverse transcriptase PCR for detection of influenza and respiratory syncytial viruses. J Clin Microbiol 53:1452–1453. doi:10.1128/JCM.03457-14. Editor: B. A. Forbes Address correspondence to Erik Munson, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03457-14
Journal of Clinical Microbiology
April 2015 Volume 53 Number 4
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olecular assays provide accurate identification of viral etiologies of influenza-like illness, potentiating expedient therapeutic decisions and/or shorter isolation stays for hospitalized patients. We report an off-label master mix modification for a commercial reverse transcriptase PCR (RT-PCR) assay that may be compatible with operations of smaller laboratories. (Results of this work were previously presented, in part, at the 114th General Meeting of the American Society for Microbiology, Boston, MA, 17 to 20 May 2014.) Prodesse ProFlu⫹ assay kits (Hologic, Inc., San Diego, CA) were obtained and stored at ⫺70°C in accordance with the manufacturer’s specifications. In an institutional review board-approved protocol, Prodesse ProFlu⫹ master mix was prepared in accordance with the assay guidelines (1) and then dispensed in 20-l aliquots into empty SmartCycler tubes that were placed in a 4°C cooling block (fresh master mix). In an experimental protocol, some of the aliquoted SmartCycler tubes were frozen at ⫺70°C for long-term storage. Prior to storage, 5.0-l aliquots of kit-provided nucleic acids were added to selected master mix tubes (positive control). Nucleic acid extracts were generated from primary nasal specimens collected in M5 MicroTest medium (Remel, Lenexa, KS) with an off-label QIAmp MinElute Virus Spin kit (Qiagen, Alameda, CA). Components of this kit were reconstituted and/or stored in accordance with the manufacturer’s specifications. Upon thawing of frozen master mix, delivery of extracted nucleic acids to the ProFlu⫹ assay was executed in accordance with the manufacturer’s specifications (1). Cycle threshold (CT) values derived from analysis of frozen control material were significantly lower than those derived from fresh controls for the time intervals studied (P ⱕ 0.0001; Table 1). Temporal-potency studies involving 40 archived extracts tested in tandem with fresh and frozen master mix yielded the expected RT-PCR amplification of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV), as well as internal control nucleic acid within extracts negative for those analytes. No significant difference between the CT values generated was noted (P ⱖ 0.22; Fig. 1). Clinical verification, utilizing 160 frozen primary clinical specimens, generated 20 nucleic acid extracts positive for influenza A virus, 16 for influenza B virus, and 20 for RSV. Tandem analysis with fresh and frozen (for 1 to 2 weeks) master mix resulted in no significant differences between CT values (P ⱖ 0.97; Fig. 2). Using data generated from fresh master mix as reference results, the initial concordance of the experimental method was 95.6%; adjudication of discrepant results by repeat testing yielded a final concordance of 98.9%. The initial indeterminate-result rate was 1.3%; this was observed in extracted specimens subjected to fresh master mix. The benefit of frozen master mix preparation, in the context of commercial DNA amplification, has been demonstrated from effi-
Letter to the Editor
FIG 2 Mean CT values obtained with nucleic acid extracts subjected to fresh (open bars) or frozen (solid bars) ProFlu⫹ master mix. Extracts were derived during a clinical verification utilizing 160 primary clinical specimens. Differences between CT values obtained with the two master mix preparations were analyzed by a t test for independent samples.
12.9 to 3.7% was observed. While master mix potency is contributory, verification of this paradigm with frozen specimens also facilitated the incorporation of a freeze-thaw step into primary specimen processing. Finally, improved laboratory practices derived from the master mix modification may include decreased technologist error, reduction of contamination risk, minimization of reagent freeze-thaw cycles, better interassay reproducibility, and an overall efficiency gain.
3.
4.
ACKNOWLEDGMENTS
5.
We are grateful to Abby Gajewski-Schwoch, Michael Brueck, and Steven Visuri for insightful discussions. B.W. and E.M. have received travel assistance from Hologic/Gen-Probe.
6.
REFERENCES
7.
1. Anonymous. 2014. Prodesse ProFlu⫹ assay product insert. Hologic, Inc., San Diego, CA. 2. Munson E, Block T, Voegeli JT, Hryciuk JE, Schell RF. 2009. Costeffective frozen master mix modification of a commercial methicillin-
April 2015 Volume 53 Number 4
resistant Staphylococcus aureus PCR assay. J Clin Microbiol 47:1888 –1891. http://dx.doi.org/10.1128/JCM.00506-09. Munson E, Kramme T, Culver A, Hryciuk JE, Schell RF. 2010. Cost-effective modification of a commercial PCR assay for detection of methicillin-resistant or -susceptible Staphylococcus aureus in positive blood cultures. J Clin Microbiol 48:1408 –1412. http://dx.doi.org/10.1128/JCM.02463-09. Munson E, Bilbo D, Paul M, Napierala M, Hryciuk JE. 2011. Modifications of commercial toxigenic Clostridium difficile PCR resulting in improved economy and workflow efficiency. J Clin Microbiol 49:2279 –2282. http://dx.doi.org/10.1128/JCM.00261-11. Moelling K. 1974. Characterization of reverse transcriptase and RNase H from friend-murine leukemia virus. Virology 62:46 –59. http://dx.doi.org /10.1016/0042-6822(74)90302-X. Das D, Georgiadis MM. 2001. A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase. Protein Sci 10:1936 –1941. http://dx.doi.org/10.1110/ps.16301. Wu D, Liu F, Liu H, Dai L, Tan D. 2014. Detection of serum HCV RNA in patients with chronic hepatitis C by transcription mediated amplification and real-time reverse transcription polymerase chain reaction. Zhong Nan Da Xue Xue Bao Yi Xue Ban 39:664 – 672. (In Chinese.) http://dx.doi .org/10.11817/j.issn.1672-7347.2014.07.003.
Journal of Clinical Microbiology
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FIG 1 Mean CT values obtained with archived nucleic acid extracts containing influenza A virus (n ⫽ 10), influenza B virus (n ⫽ 10), RSV (n ⫽ 10), or internal control RNA (n ⫽ 10) that were subjected to fresh ProFlu⫹ master mix (0 week) and master mix frozen for 2, 4, 8, or 13 weeks. Temporal differences in master mix potency for each analyte were assessed by one-way analysis of variance.
Frozen Master Mix-Based Modification of Commercial Reverse Transcriptase PCR for Detection of Influenza and Respiratory Syncytial Viruses Brooke Wenninger,a Erik Munson,a,b Maureen Napierala,a Kimber L. Munson,a Dorothy Bilbo,a Jeanne E. Hryciuka Wheaton Franciscan Laboratory, Milwaukee, Wisconsin, USAa; College of Health Sciences, University of Wisconsin—Milwaukee, Milwaukee, Wisconsin, USAb
M
1452
jcm.asm.org
TABLE 1 Mean CT values obtained with replicates of prepared ProFlu⫹ positive-control material frozen for 2, 4, 8, or 13 weeks at ⫺70°Ca Mean CT value obtained after storage for: Control Influenza A virus Influenza B virus RSV A RSV B
0 wkb c
29.79 30.08c 28.88c 29.79c
2 wk
4 wk
8 wk
13 wk
29.14 28.03 27.71 29.47
28.83 27.82 27.54 29.40
28.88 27.89 27.25 29.17
29.20 28.15 27.90 29.64
a Temporal differences in master mix potency for each control were assessed by oneway analysis of variance. b Fresh master mix. c Fresh master mix P ⱕ 0.0001 versus all intervals of master mix freezing.
cacy (2, 3) and workflow (4) perspectives. Within the ProFlu⫹ assay, the catalyst for nucleic acid amplification is Moloney murine leukemia virus (MMLV) RT (5). The molecule itself is labile; i.e., nonionic detergents and glycerol are requirements for in vitro solubility (6). Package insert data emphasize that the master mix (components of which include separately packaged MMLV RT and an RNase inhibitor) must be prepared fresh for each RT-PCR run (1). However, the experiments presented demonstrate that prolonged frozen storage of prepared ProFlu⫹ control and master mix reagents at ⫺70°C yielded accurate results; in some instances, potency greater than that obtained with fresh master mix was observed. A paucity of literature exists with respect to extended stability of frozen MMLV RT; one report in the Chinese literature (7) suggests that a laboratory-developed RNA amplification assay exhibited appropriate stability when reagents were stored for up to 6 months at ⫺20°C. Furthermore, small-scale experiments not presented in this report reveal that frozen ProFlu⫹ master mix and controls possess sufficient potency following 12 to 15 months of storage at ⫺70°C. Pending in-house verification studies, these data provide laboratories an additional option for accurate and efficient detection of respiratory virus infection agents. Following introduction of the verified frozen master mix paradigm into routine laboratory practice, a reduction of average indeterminate-result rates from
Accepted manuscript posted online 28 January 2015 Citation Wenninger B, Munson E, Napierala M, Munson KL, Bilbo D, Hryciuk JE. 2015. Frozen master mix-based modification of commercial reverse transcriptase PCR for detection of influenza and respiratory syncytial viruses. J Clin Microbiol 53:1452–1453. doi:10.1128/JCM.03457-14. Editor: B. A. Forbes Address correspondence to Erik Munson, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03457-14
Journal of Clinical Microbiology
April 2015 Volume 53 Number 4
Downloaded from http://jcm.asm.org/ on November 15, 2015 by guest
olecular assays provide accurate identification of viral etiologies of influenza-like illness, potentiating expedient therapeutic decisions and/or shorter isolation stays for hospitalized patients. We report an off-label master mix modification for a commercial reverse transcriptase PCR (RT-PCR) assay that may be compatible with operations of smaller laboratories. (Results of this work were previously presented, in part, at the 114th General Meeting of the American Society for Microbiology, Boston, MA, 17 to 20 May 2014.) Prodesse ProFlu⫹ assay kits (Hologic, Inc., San Diego, CA) were obtained and stored at ⫺70°C in accordance with the manufacturer’s specifications. In an institutional review board-approved protocol, Prodesse ProFlu⫹ master mix was prepared in accordance with the assay guidelines (1) and then dispensed in 20-l aliquots into empty SmartCycler tubes that were placed in a 4°C cooling block (fresh master mix). In an experimental protocol, some of the aliquoted SmartCycler tubes were frozen at ⫺70°C for long-term storage. Prior to storage, 5.0-l aliquots of kit-provided nucleic acids were added to selected master mix tubes (positive control). Nucleic acid extracts were generated from primary nasal specimens collected in M5 MicroTest medium (Remel, Lenexa, KS) with an off-label QIAmp MinElute Virus Spin kit (Qiagen, Alameda, CA). Components of this kit were reconstituted and/or stored in accordance with the manufacturer’s specifications. Upon thawing of frozen master mix, delivery of extracted nucleic acids to the ProFlu⫹ assay was executed in accordance with the manufacturer’s specifications (1). Cycle threshold (CT) values derived from analysis of frozen control material were significantly lower than those derived from fresh controls for the time intervals studied (P ⱕ 0.0001; Table 1). Temporal-potency studies involving 40 archived extracts tested in tandem with fresh and frozen master mix yielded the expected RT-PCR amplification of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV), as well as internal control nucleic acid within extracts negative for those analytes. No significant difference between the CT values generated was noted (P ⱖ 0.22; Fig. 1). Clinical verification, utilizing 160 frozen primary clinical specimens, generated 20 nucleic acid extracts positive for influenza A virus, 16 for influenza B virus, and 20 for RSV. Tandem analysis with fresh and frozen (for 1 to 2 weeks) master mix resulted in no significant differences between CT values (P ⱖ 0.97; Fig. 2). Using data generated from fresh master mix as reference results, the initial concordance of the experimental method was 95.6%; adjudication of discrepant results by repeat testing yielded a final concordance of 98.9%. The initial indeterminate-result rate was 1.3%; this was observed in extracted specimens subjected to fresh master mix. The benefit of frozen master mix preparation, in the context of commercial DNA amplification, has been demonstrated from effi-
Letter to the Editor
FIG 2 Mean CT values obtained with nucleic acid extracts subjected to fresh (open bars) or frozen (solid bars) ProFlu⫹ master mix. Extracts were derived during a clinical verification utilizing 160 primary clinical specimens. Differences between CT values obtained with the two master mix preparations were analyzed by a t test for independent samples.
12.9 to 3.7% was observed. While master mix potency is contributory, verification of this paradigm with frozen specimens also facilitated the incorporation of a freeze-thaw step into primary specimen processing. Finally, improved laboratory practices derived from the master mix modification may include decreased technologist error, reduction of contamination risk, minimization of reagent freeze-thaw cycles, better interassay reproducibility, and an overall efficiency gain.
3.
4.
ACKNOWLEDGMENTS
5.
We are grateful to Abby Gajewski-Schwoch, Michael Brueck, and Steven Visuri for insightful discussions. B.W. and E.M. have received travel assistance from Hologic/Gen-Probe.
6.
REFERENCES
7.
1. Anonymous. 2014. Prodesse ProFlu⫹ assay product insert. Hologic, Inc., San Diego, CA. 2. Munson E, Block T, Voegeli JT, Hryciuk JE, Schell RF. 2009. Costeffective frozen master mix modification of a commercial methicillin-
April 2015 Volume 53 Number 4
resistant Staphylococcus aureus PCR assay. J Clin Microbiol 47:1888 –1891. http://dx.doi.org/10.1128/JCM.00506-09. Munson E, Kramme T, Culver A, Hryciuk JE, Schell RF. 2010. Cost-effective modification of a commercial PCR assay for detection of methicillin-resistant or -susceptible Staphylococcus aureus in positive blood cultures. J Clin Microbiol 48:1408 –1412. http://dx.doi.org/10.1128/JCM.02463-09. Munson E, Bilbo D, Paul M, Napierala M, Hryciuk JE. 2011. Modifications of commercial toxigenic Clostridium difficile PCR resulting in improved economy and workflow efficiency. J Clin Microbiol 49:2279 –2282. http://dx.doi.org/10.1128/JCM.00261-11. Moelling K. 1974. Characterization of reverse transcriptase and RNase H from friend-murine leukemia virus. Virology 62:46 –59. http://dx.doi.org /10.1016/0042-6822(74)90302-X. Das D, Georgiadis MM. 2001. A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase. Protein Sci 10:1936 –1941. http://dx.doi.org/10.1110/ps.16301. Wu D, Liu F, Liu H, Dai L, Tan D. 2014. Detection of serum HCV RNA in patients with chronic hepatitis C by transcription mediated amplification and real-time reverse transcription polymerase chain reaction. Zhong Nan Da Xue Xue Bao Yi Xue Ban 39:664 – 672. (In Chinese.) http://dx.doi .org/10.11817/j.issn.1672-7347.2014.07.003.
Journal of Clinical Microbiology
jcm.asm.org
1453
Downloaded from http://jcm.asm.org/ on November 15, 2015 by guest
FIG 1 Mean CT values obtained with archived nucleic acid extracts containing influenza A virus (n ⫽ 10), influenza B virus (n ⫽ 10), RSV (n ⫽ 10), or internal control RNA (n ⫽ 10) that were subjected to fresh ProFlu⫹ master mix (0 week) and master mix frozen for 2, 4, 8, or 13 weeks. Temporal differences in master mix potency for each analyte were assessed by one-way analysis of variance.
