Second-generation technologies in molecular microbiology

15. Kerr, R.A. 1994. Climate modeling's fudge factor comes under fire. Science 265:1528.

encephalitis viruses by Culex tarsalis (Diptera: Culicidae). J. Med. Entomol. 30:151-160. 24. Frier, J.E. 1993. Eastern equine encephalomyelitis. Lancet 342:1281-1282. 25. Centers for Disease Control and Prevention. 1998. Imported dengue - - United States, 1996. MMWR 47:544-547.

16. Karl, T.R., R.W. Knight, and N. Plummer. 1995. Trends in high-frequency climate variability in the twentieth century. Nature 377:217-220.

26. Centers for Disease Control and Prevention. 1996. Dengue fever at the U.S.-Mexico border, 1995-1996. MMWR 45:841-844.

17. Stone, R. 1995. If the mercury soars, so may the health hazards. Science 267:957-958.

27. Centers for Disease Control and Prevention. 1989. Update: Aedes albopictus infestation - - United States, Mexico. MMWR 38:440-446.

13. Epstein, P.R. and D. Sharp. 1993. Commentary: medicine in a warmer world. Lancet 342:1004. 14. Wigley, T.M.L. 1995. A successful prediction? Nature 376:463-464.

18. Reeves, W.C. et al. 1994. Potential effect of global warming on mosquito-borne arbovimses. J. Med. Entomol. 31:323-332. 19. Rueda L.M. et al. 1990. Temperaturedependent development and survival rates of Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 27:892-898. 20. Rogers, D.J. and M.J. Packer. 1993. Vector-bome diseases, models, and global climate change. Lancet 342:1282-1284. 21. Watts, D.M. et al. 1987. Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus. Am. J. Trop. Med. Hyg. 36:143-152. 22. Noden, B.H., M.D. Kent, and J.C. Beier. 1995. The impact of variations in temperature on early Plasmodium falciparum development in Anopheles stephensi. Parasitology 111:539-545. 23. Reisen, W.K. et al. 1993. Effect of temperature on the transmission of western equine encephalomyelitis and St. Louis

28. Moore, C.G. and C.J. Mitchell. 1997. Aedes albopictus in the United States: ten year presence and public health implications. Emerg. Infect. Dis. 3:329-334. 29. Monath, T.P. and T.F. Tsai. 1987. St. Louis encephalitis: lessons from the last decade. Am. J. Trop. Med. Hyg. 37 Suppl:40S-59S. 30. Herrera-Basto, E. et al. 1992. First reported outbreak of classical dengue fever at 1700 meters above sea level in Guerrero State, Mexico, June 1988. Am. J. Trop. Med. Hyg. 46:649-653. 31. Loevinsohn, M.E. 1994. Climatic warming and increased malaria incidence in Rwanda. Lancet 343:714-718. 32. Nicholls, N. 1993. E1 Nifio-Southern Oscillation and vector-borne disease. Lancet 342:1284-1285. 33. http://www.pmel.noaa.gov (National Oceanic and Atmospheric Administration website).

34. Manabe, S. and R.J. Stouffer. 1993. Century scale effects of increased atmospheric CO2 on the ocean-atmosphere system. Nature 364:215-218. 35. Epstein, P.R. 1995. Emerging diseases and ecosystem instability: new threats to public health. Am. J. Public Health 85:168-172. 36. Epstein, P.R., T.E. Ford, and R.R. Colwell. 1993. Marine ecosystems. Lancet 342:1216-1219. 37. Huq, A. et al. 1984. The role of planktonic copepods in the survival and multiplication of lqbrio cholerae in the aquatic environment, p.521-534. In R.R. Colwell (ed.), Vibrios in the environment. John Wiley & Sons, Inc., New York. 38. Huq, A. et al. 1990. Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods. Appl. Environ. Microbiol. 56:2370-2373. 39. Colwell, R.R. 1996. Global climate and infectious disease: the cholera paradigm. Science 274:2025-2031. 40. Washino, R.K. and B.L. Wood. 1994. Application of remote sensing to arthropod vector surveillance and control. Am. J. Trop. Med. Hyg. 50 Suppl:134-144. 41. http://geo.arc.nasa.gov (National Aeronautics and Space Administration website). 42. Epstein, P.R., D.J. Rogers, and R. Slooff. 1993. Satellite imaging and vector-borne disease. Lancet 341:1404-1406. 43. Rogers, D.J. and S.E. Randolph. 1991. Mortality rates and population density of tsetse flies correlated with satellite imagery. Nature 351:739-741.

Editorial

Second-Generation Technologies in Molecular Microbiology John B. Watson, Ph.D. Director of Molecular Diagnostics Epicentre Technologies Madison, WI 53713

Introduction The last five years have seen a dramatic increase in the use of D N A -and R N A - b a s e d methods for the identification and typing o f microorganisms in the clinical m i c r o b i o l o g y laboratory. In fact, the change has been so rapid that many clinical laboratory personnel have had a difficult time mastering the new molecular technologies. In this article, I will describe some o f the methods that

86

0196-4399/0o (seefrontmatter)

have been developed to ease the transition o f nucleic acid-based techniques from research laboratories into diagnostic laboratories. These include "secondgeneration" molecular methods specifically designed to be used in the clinical laboratory by personnel trained in medical technology rather than molecular biology.

First-Generatlon Molecular Methods To understand the forces driving the development o f second-generation technologies, it is important to describe why

© 2000ElsevierScienceInc.

first-generation technologies fail to meet the needs of clinical laboratories. Many molecular methods were developed during the 1960s and 1970s in biochemistry laboratories where hazardous chemicals and complicated protocols were not considered barriers to method development (1). One of the most c o m m o n methods in molecular biology uses phenol and chloroform for the extraction of DNA, and a standard method for purifying R N A uses the hazardous chemical guanidinium thiocyanate along with phenol. Not only do these methods use hazardous reagents, but each method

ClinicalMicrobiologyNewsletter22:11.2000

uses substantially different protocols so that clinical laboratories need to invest substantial resources in training personnel to be competent in extracting nucleic acids from the various materials received by clinical laboratories. After the invention of PCR and the subsequent application of molecular methods to clinical questions, the field was slow in developing new protocols appropriate to diagnostic laboratories (1).

1) Make a Mastermix containing enzyme, template and primers

Second-Generation Reagents.

m

A

B ¢ D E F G H I (PCR PreMix Buffer)

J

K

L

2) Run 12 PCR reactions using a 1:1 mixture of the 12 MasterAmp PreMix and the Mastermix

4PCR

Fragment A BCDFG

H I

J K L

(PCR PreMix Buffer) 3) Select the MasterAmp PCR PreMix buffer that provides the best amplification

G G G G G G G GG

For example, most PCR assays still require 6 to 10 separate pipetting steps to prepare a single sample. Each step introduces another variable into the reaction resulting in unacceptable lab-tolab and day-to-day variability in PCR. This is of minor concern to research laboratories where it is often relatively trivial to rerun an experiment. In clinical laboratories, PCR variability has been a major factor in the slow acceptance of molecular techniques in the clinical microbiology laboratory and has resulted in many clinicians underutilizing molecular assays. It is into this vacuum that companies have attempted to place methodologies that (i) eliminate the use of hazardous reagents; (ii) improve day-to-day reproducibility of molecular methods; and (iii) reduce the cost and time necessary to develop molecular methods. One example is the GEN-ETI-K" DEIA (3) from DiaSorin which detects doublestranded DNA formed by the hybridization of target DNA with a specific DNA probe by using a monoclonal antibody against double-stranded DNA. In practice, this involves the development of specific PCR primers and the detection of the amplified product using a probe designed by the manufacturer. In some cases, custom designed kits can be developed for individual customers. This is clearly a second-generation technology in that it significantly reduces the effort needed by the investigator to develop the detection system for a PCR assay; yet, it falls short of third-generation technologies that must undergo complete FDA review and are complete kits from extraction to interpretation. As a second-generation assay, the

Cells

Tissue

White Cells

I

I

I

I

WWW-

Bef/y Cost

Lyselcells (poe-treat with RNase if necessary)

W

Precipitate and spin down proteins

W

Precipitate and wash DNA or total nucleic acid

W

Rasuspend DNA or total nucleic acid

W-W

For UltraPure RNA (I) Treat with DNase (ii) PrecipitateDNase 0il) Precipitate, wash & rasuspend RNA

Figure 2. A second-generation protocol for DNA and~or RNA purification. The MasterPure DNA and RNA Purification Kit is designed to extract DNA, RNA, or total nucleic acid in less than an hour The kit has been used to isolate viral bacterial fungaL and mammalian nucleic acids from blood, urine, saliva, and many other biological materials.

GEN-ETI-K DEIA kit has the advantage of being more flexible and less expensive than third-generation assays. Second-generation reagents have also been developed for PCR buffer systems. These include the AmershamPharmacia Ready-To-Go'" PCR beads and Epicentre's MasterAmp PCR PreMixes. Both kits provide all of the buffer components necessary for PCR amplification. The major advantage of these buffers is that they eliminate the day-to-day variability of PCR by eliminating most pipetting steps needed to set up a reaction. In addition, TM

Figure 3. A highly sensitive method for extraction of nucleic acids. The MasterPure DNA and RNA Purification Kit was used to isolate DNA from 100 pl of serial dilutions of an Escherichia coli culture. The number ofE. coli cells extracted in lanes 1-6, respectively, were 2 x 107, 2x106, 2x105, 2x104, 2,000, and 200 ; lane 7 is the negative control.

G G G

(PCR PreMix Buffer) Figure 1. Protocol for PCR optimization by using the MasterAmp PCR Optimization Kit. After performing the PCR reaction with 12 PCR PreMixes that vary in Mg +÷ concentration and MasterAmp PCR enhancer concentration, one of the buffers is selected for use in future amplifications.

Clinical Microbiology Newsletter 22:11,2000

Liquid

© 2000 Elsevier Science Inc.

0196-4399/00 (see frontmatter)

87

the MasterAmp kits include a PCR enhancer which contains betaine. This enhancer has been shown to improve the consistency of almost any PCR reaction. In particular, the MasterAmp Enhancer improves amplifications of templates that are high in GC content, longer than 2 kb, multiplex amplifications, and/or in samples containing small amounts of starting sample. The protocol involves selecting the optimal PCR PreMix from the 12 PreMixes in the kit and then using only that PreMix for consistency in subsequent amplifications (Figure 1). The importance of consistent PCR results in clinical laboratories and clinical research laboratories can not be overstated. The expense of repeating an assay due to random variability in controls can run into thousands of dollars per assay for personnel and reagent costs. Another source of variation in molecular assays is in the extraction of the nucleic acids. First-generation methodologies not only use hazardous reagents, but they also suffer from variability in reagent lot quality, complicated protocols, and results that vary with operator skill. Spin column methods for DNA and RNA purification address some of the problems with first-generation methods in that they eliminate hazard-

Editors:

ous reagents and provide reagents that have quality specifications. Unfortunately, spin columns introduce other difficulties such as the production of large amounts of plastic waste and cumbersome protocols that require training to perform consistently. The PureGene system from Gentra was the first commercially-available kit to use the extremely simple and non-hazardous salt precipitation method of Miller et al. (4) for the purification of DNA. The MasterPure Complete DNA and RNA Purification Kit from Epicentre improves on the Miller protocol by incorporating a co-precipitant into the kit, which uses the same simple protocol (Figure 2), but can be used to purify DNA and/or RNA from virtually any sample even those that contain extremely small numbers of organisms (Figure 3). This method allows clinical laboratories to use a single protocol for purification of nucleic acids from bacteria, RNA viruses, DNA viruses, and fungi found in urine, blood, CSF, saliva, and other clinical specimens (5). TM

TM

Conclusion By using second-generation technologies for the extraction, amplification, and detection of nucleic acids, clinical laboratories can reduce molecular assay

method development times from months to days. This allows laboratories to offer tests at a cost that will encourage increased physician utilization. Once molecular tests are being routinely ordered by clinicians, laboratories will feel comfortable expanding their test menus. It is hoped that second-generation technologies will help encourage the use of molecular assays in clinical microbiology laboratories. References

1. Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor, Cold Spring Harbor Laboratory. 2. Miller, S.A., D.D. Dykes, and H.E Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research 16:1215. 3. Rutledge, B.J. Mayo Clinic, Rochester, MN. Comparative evaluation of colorimetric microplate systems for identification of amplicon specific for HSV in CSE 14th Annual Clinical Virology Symposium, 1998. 4. Saiki, R. et al. 1985. Enzymatic amplification of 13-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350. 5. Watson, J.B. et al. 1998. A new method for DNA and RNA purification. J. Clin. Ligand Assay 21:394.

General Information

Mary Jane Ferraro Paul A. Granato Josephine A. Morello R.J. Zabransky © 2000 Elsevier Science Inc. ISSN 0196-4399 CMNEEJ 22(11)81-88, 2000 Elsevier

Subscription information can be found inside the front cover. This newsletter has been registered with the Copyright Clearance Center, Inc. Consent is given for copying articles for personal or internal use, or for the personal or internal use of specific clients. This consent is given on the condition that the copier pay through the Center the per-page fee stated in the code on the first page for copying beyond that permitted by the US Copyright Law. If no code appears on an article, the author has not given broad consent to copy and permission to copy must be obtained directly from the author. This consent does not extend to other kinds of copying, such as for general distribution, resale, advertising and promotional purposes, or for creating new collective works. Address orders, changes of address, and claims for missing issues to Journal Fulfillment Department, Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Claims for missing issues can be honored only up to three months for domestic addresses and six months for foreign addresses. Duplicate copies will not be sent to replace ones undelivered due to failure to notify Elsevier of change of address. Postmaster: Send address changes to Clinical Microbiology Newsletter, Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Editorials and letters printed in this newsletter are published for the interest of the readers and do not necessarily reflect the opinions of the editors.

Clinical Microbiology Newsletter is abstracted in Tropical Diseases Bulletin. Abstracts on Hygiene and Communicable Diseases, EMBASE/Excerpta Medica, and Current AIDS Literature.

IllHllillLIIIIIIII]i1111111111111 0196-4399(20000601 )22:11; 1 -G

88

0196-4399100 (see frontmatter)

Full text of this and previous issues can viewed online using the ScienceDirect or ScienceDirect Web-Editions services. Visit http://www.sciencedirect.com or http://www.web-editions.com for more details and access.

© 2000 Elsevier Science Inc.

Clinical Microbiology Newsletter 22:11,2000