JCM Accepted Manuscript Posted Online 28 January 2015 J. Clin. Microbiol. doi:10.1128/jcm.03457-14 Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 Frozen Master Mix Modification of Commercial Reverse-transcriptase PCR for Detection of Influenza and Respiratory Syncytial Viruses 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Brooke Wenninger, 1 Erik Munson, 1,2 * Maureen Napierala, 1 Kimber L. Munson, 1 Dorothy Bilbo, 1 and Jeanne E. Hryciuk 1 Wheaton Franciscan Laboratory, 1 Milwaukee, Wisconsin 53215; and College of Health Sciences, University of Wisconsin--Milwaukee, 2 Milwaukee, Wisconsin 53201 Running title: New Data Letter * -- Corresponding author Erik Munson Wheaton Franciscan Laboratory St. Francis Hospital 3237 South 16th Street Milwaukee, WI 53215 Telephone: (414) 647-7589 Facsimile: (414) 647-5504 21 22 Electronic mail: Erik.Munson@wfhc.org 23 24 Results of this work were previously presented, in part, at the 114th General Meeting of the American Society for Microbiology, Boston, Mass., 17-20 May 2014.
2 25 26 27 28 Molecular 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 assay (RT-PCR) that may be compatible with operations of smaller laboratories. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Prodesse ProFlu+ assay kits (Hologic, San Diego, CA) were obtained and stored at -70 C per manufacturer specifications. In an Institutional Review Board-approved protocol, Prodesse ProFlu+ master mix was prepared per assay guidelines [1], then dispensed in 20-μL aliquots to 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 longterm storage. Prior to storage, 5.0-μL 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 media (Remel, Lenexa, KS) with an off-label QIAmp MinElute Virus Spin Kit (Qiagen, Alameda, CA). Components of this kit were reconstituted and/or stored per manufacturer specifications. Upon thawing of frozen master mix, delivery of extracted nucleic acids to the ProFlu+ assay was executed per manufacturer specifications [1]. Cycle threshold (C T ) values derived from analysis of frozen control material were significantly lower when compared to fresh controls for the time intervals studied (P 0.0001; Table 1). Temporal potency studies, involving 40 archived extracts tested in tandem with fresh and frozen master mix, yielded expected RT-PCR amplification of influenza A, influenza B, and respiratory
3 47 48 syncytial viruses (RSV), as well as internal control nucleic acid within extracts negative for those analytes. No significant difference was noted between generated C T values (P 0.22; Fig 1). 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 Clinical verification, utilizing 160 frozen primary clinical specimens, generated 20 nucleic acid extracts positive for influenza A, 16 for influenza B, and 20 for RSV. Tandem analysis with fresh and frozen (1- to 2-week) master mix resulted in no significant differences between C T values (P 0.97; Fig. 2). Using data generated from fresh master mix as reference results, 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 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 efficacy [2,3] and workflow [4] perspectives. Within the ProFlu+ assay, the catalyst for nucleic acid amplification is Moloney murine leukemia virus reverse transcriptase (MMLV-RT) [5]. The molecule itself is labile; i.e., non-ionic detergents and glycerol are requirements for in vitro solubility [6]. Package insert data emphasize that 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 presented experiments demonstrate that prolonged -70 C frozen storage of prepared ProFlu+ control and master mix reagents yielded accurate results; in some instances, greater potency was observed when compared to utilization of fresh master mix. A paucity of literature exists with respect to extended frozen stability of MMLV-RT; one report in the Chinese literature [7] suggests that a laboratory-developed RNA amplification assay exhibited appropriate stability when reagents
4 70 71 72 were stored 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 12-15 months of -70 C storage. 73 74 75 76 77 78 79 80 81 82 83 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 rates from 12.9% to 3.7% was observed. While master mix potency is contributory, verification of this paradigm using frozen specimens also facilitated incorporation of a freeze/thaw step into routine 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, inter-assay reproducibility, and overall efficiency gain.
5 84 ACKNOWLEDGMENTS The authors 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.
85 REFERENCES 86 87 1. Prodesse ProFlu+ product insert. 2014. Hologic, Inc., San Diego, California. 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 2. Munson, E., T. Block, J. T. Voegeli, J. E. Hryciuk, and R. F. Schell. 2009. Costeffective frozen master mix modification of a commercial methicillin-resistant Staphylococcus aureus PCR assay. J. Clin. Microbiol. 47: 1888-1891. 3. Munson, E., T. Kramme, A. Culver, J. E. Hryciuk, and R. F. Schell. 2010. Costeffective 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. 4. Munson, E., D. Bilbo, M. Paul, M. Napierala, and J. E. Hryciuk. 2011. Modifications of commercial toxigenic Clostridium difficile PCR resulting in improved economy and workflow efficiency. J. Clin. Microbiol. 49: 2279-2282. 5. Moelling, K. 1974. Characterization of reverse transcriptase and RNase H from friendmurine leukemia virus. Virology 62: 46-59. 6. Das, D., and M. M. Georgiadis. 2001. A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase. Protein Sci. 10: 1936-41.
7 108 109 110 111 7. Wu, D., F. Liu, H. Liu, L. Dai, and D. Tan. 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: 664-72. (Chinese) 112 113 114 Downloaded from http://jcm.asm.org/ on April 13, 2018 by guest
115 116 117 TABLE 1: Mean cycle threshold values for replicates of prepared ProFlu+ positive control material frozen 2, 4, 8, and 13 weeks at -70 C. Temporal differences in master mix potency for each control were assessed by one-way analysis of variance. 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 Control Cycle Threshold per Interval of Storage 0 week 2 weeks 4 weeks 8 weeks 13 weeks Influenza A 29.79 29.14 28.83 28.88 29.20 Influenza B 30.08 28.03 27.82 27.89 28.15 RSV A 28.88 27.71 27.54 27.25 27.90 RSV B 29.79 29.47 29.40 29.17 29.64 Fresh master mix Fresh master mix P 0.0001 versus all intervals of frozen master mix
9 145 146 147 148 149 Figure 1. Mean cycle threshold values for archived nucleic acid extracts containing influenza A (n = 10), influenza B (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, and 13 weeks. Temporal differences in master mix potency for each analyte were assessed by one-way analysis of variance. 150 151 Downloaded from http://jcm.asm.org/ on April 13, 2018 by guest
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11 153 154 Figure 2. Mean cycle threshold values for nucleic acid extracts subjected to fresh (open bars) and frozen (solid bars) ProFlu+ master mix; extracts were derived during a 155 clinical verification utilizing 160 primary clinical specimens. Differences 156 157 between cycle threshold values derived from the two master mix preparations were analyzed by the t-test for independent samples. 158 Downloaded from http://jcm.asm.org/ on April 13, 2018 by guest
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Cycle Threshold 34 33 32 31 30 29 28 27 26 25 24 P = 0.22 P = 0.99 P = 0.99 P = 1.00 Influenza A Influenza B RSV Internal control Analyte 0 week 2 weeks 4 weeks 8 weeks 13 weeks
Cycle Threshold 30 29 28 27 26 25 24 23 P = 0.98 P = 1.00 P = 0.97 Influenza A Influenza B RSV Analyte