Oral Microbiot Itnmwtot 1989: 4: 141-145
`
`"Reverse" DNA hybridization
`method for the rapid identification
`of subgingivai microorganisms
`
`Smith GEE, Socrattstcy SS, Satisotie C. "Reverse" DNA hybridization method for
`the rapid identification of subgingival microorganisms.
`Orat Microbiot Imtnunot 1989: 4: 141-145.
`
`A "reverse" hybridization method is described, in which whole chromosomal
`DNA was extracted from 10-20 colonies of "unknown" strains in pure culture
`and labelled with digoxigenin by a random primer teehnique. DNA probes were
`prepared from a total of 23 strains and hybridized with targets containing 100
`ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose.
`21/23 digoxigenin-labelled DNA probes successfully detected all members of the
`homologous species present on filters. Probes to Eusobactcriwn nucleattitn
`strains 364 and MG detected 3/4 and 1/4 members of this speeies, respectively;
`13/23 probes were 100% specific, but cross reactions between 10 probes and DNA
`targets from closely related, heterologous species occurred in 15/834 possible
`instances. False-positive reactions that occurred between closely related speeies
`were, however, easily distinguished and did not prevent the aecurate identification
`of probe stiains. Digoxigenin-labelled probes were capable of detecting 100 pg of
`homologous DNA. The reverse hybridization procedure allows identification or
`grouping of a large number of isolates within 3 days and provides a more
`economieal means of characterizing subgingival isolates than predominant culti-
`vable techniques and conventional phenotypic testing. This method could be
`adapted for the direct identifieation of microorganisms in subgingival plaque
`samples
`
`G. L. F. Smith, S. S. Socransky,
`C. Sansone
`Forsyth Dental Center, Boston, USA
`
`Key words: subgingival microorganisms: re-
`verse hybridization: digoxigenin-labelled DNA
`probes.
`Dr. Gillian Smith, Forsyth Dental Center, 140
`The Fenway, Boston, MA 02115, USA.
`Accepted (or publication February 15, 1989
`
`The identification of pure cultures from
`subgingival plaque samples is often a
`time-consuming, labor-intensivepioeess
`requiring specialized laboratory tech-
`niques and considerable expertise. Even
`when these ideal eonditions prevail,
`problems such as the failure of fastidi-
`ous organisms to grow in available me-
`dia, a paucity of well-defined pheno-
`typic markers for many species, or
`variability in test results may contribute
`to equivocal identifieations. Attempts
`have been made to overcome these diffi-
`culties using DNA probes as an adjunct
`to, or a replacement for, conventional
`characterization (5, 7, 8). The classical
`approach to DNA probe identifications
`is to place cells of the test species on a
`solid support, such as nitrocellulose,
`and use a series of treatments to lyse
`the cells, denature and bind the released
`DNA to the solid support (3, 10). Filters
`carrying the test organisms' DNAs are
`hybridized with probes constructed
`
`from purified referenee strain DNA. Al-
`though it has a number of advantages,
`this procedure also has several draw-
`baek. Unless the investigator has a
`reasonable idea of the identity of the
`test species, the number of filters pre-
`pared and probes employed could be
`quite large before the organism is sue-
`cessfully identified. Furthermore, in its
`elassical form, the teehnique is not well
`suited to the identification of small
`numbers of isolates, sinee the work in-
`volved in preparing and hybridizing 96
`unidentified stiains on a filter is com-
`parable to that involved in identifying
`a single strain by the same method. In
`sueh situations, the investigator may
`choose to wait until sufficient strains
`have aecumulated to make filter prep-
`aration and hybridizations worthwhile.
`A more practical approach to strain
`identification would be to prepare a
`single strain for hybridization in sueh a
`way that it could be simultaneously
`
`tested against virtually any number of
`reference species DNAs. To achieve this
`goal, we investigated the concept of "re-
`verse" hybridization, in which a small
`number of cells of "unknown" test spe-
`cies (in pure culture) were treated to
`release, denature and label their DNAs
`with digoxigenin. Thus, the unknown
`isolate became the probe, which was
`then hybridized with a range of purified,
`denatured reference strain DNAs fixed
`to nitrocellulose. In this way, unknown
`isolates could be identified or grouped
`by positive reaetions appearing at ap-
`propriate sites on the filters. Previous
`experiments (unpublished observations)
`indicated that inclusion of hexadecyltri-
`methyl ammonium bromide (CTAB; 11)
`in the extraction of DNA from 10* cells
`produced DNA which could be biotin-
`labelled by a random primer teehnique
`(2). The present study examined the
`possibility of using this technique to
`prepare digoxigenin-labelled probes
`
`Enzo Exhibit 2016
`BD v. Enzo
`Case IPR2017-00172
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`142
`
`Smith et at.
`
`with DNA extracted from small num-
`bers of cells in the same manner.
`
`Material and methods
`Sources and cultivation ot strains
`Thirty-three gram negative and 5 gram
`positive strains were used in this study
`(Table 1). Strains were grown on Tryp-
`ticase soy agar plates supplemented
`with 5% sheep blood (Baltimore Bio-
`logical Laboratories, Cockeysville,
`MD), in an atmosphere of 80% N2,
`10% H2 and 10% CO^ at 35°C. Gram
`negative strains were incubated for 3-5
`days and gram positive organisms for
`1-2 days; plates inoculated with Bac-
`teroides forsythus were incubated for
`5-7 days in parallel with a supporting
`strain of Eusobacterium nueteatum
`
`(FDC 364). Strains belonging to the
`species Wolinelta, Campytobacter and
`Bacteroides gracitis were grown on
`brain heart infusion agar (Baltimore
`Biological Laboratories), supplemented
`with 0.2% sodium formate, 0.3% diso-
`dium fumarate and 1 % hemin.
`
`"spot" contained 100 ng of purified tar-
`get DNA. Filters were soaked for 10
`min in 0.5 M NaOH, 1.5 M NaCl
`(lysing/denaturing
`solution), blotted
`dry, and then soaked in 1.5 M NaCl,
`0,5 M Tris-HCl, pH 7.6 (neutralizing
`solution). After drying in air for 15-20
`min, filters were baked at 80"C for 2 h.
`
`Preparation of filters
`Confluent growth was removed from
`the surface of 1-2 agar plates and test
`strain DNAs were purified as previously
`described (9). Aliquots of the 38 DNA
`preparations were placed in the wells of
`microtiter plates (see Fig. 1 legend) and
`transferred to nitrocellulose (BA 85,
`0.45 fixn, Schleicher & Schuell, Inc.,
`Keene, NH) using an MIC inoculator
`(Dynateeh Inc., Alexandria, VA.). Each
`
`Extraction of DNA for probe preparation
`
`A modified version of the method for
`DNA purification deseribed above was
`used to extract chromosomal DNA
`from a small number of cells that was
`suitable for digoxigenin labeling. In
`brief, 10-20 colonies were scraped from
`the surface of agar plates and resus-
`pended in 557 /il TE (10 mM Tris, pH
`7.6, 1 mM EDTA). In addition, I /<g of
`
`Tatite I. List of strains which had target DNAs fixed to nitrocellulose.
`
`Species
`Streptoeoeeus sanguis I
`Streptoeoeeus sanguis II
`Streptoeoeeus intermedius
`Peptostreptoeoeeus mieros
`Aetinomyees naestundii
`Baeteroides veroratis
`Baeteroides graeitis
`Baeteroides graeilis
`Baeteroides graeitis
`Baeteroides intermedius
`Baeteroides intermedius
`Baeteroides intermedius
`Baeteroides gingivatis
`Baeteroides forsythus
`Wotinetta eurva
`Wotinella curva
`Wolinella eurva
`Wolinelta sputigena
`Wotinella reeta
`Wotinetta recta
`Capnoeytophaga sputigena
`Capnoeytophaga oehraeea
`Capnoeytophaga oehraeea
`Capnocytoptiaga gingivatis
`Capnoeytophaga type IV
`Eikenetta eorrodens
`Campylohaeter eoneisus
`Baeteroides zoogteoformans
`Baeteroides heparinotytieus
`Veillonelta parvuta
`Fusobaeterium nueteatum
`Fusobaeterium nueteatum
`Fusobaeterium nueleatum
`Eusobaeterium nueleatum
`Aetino. aetinomyeetemeomitans
`Actino. actinomycetemcomitans
`Haemophilus aphrophilus
`Haemophilus aphrophilus
`
`Strain*
`
`F C OI
`F Al
`F JS26
`F JH20
`A 12104
`A 33779
`F 402
`F 1083
`F 406
`F 581
`V 8944
`A 25261
`F 381
`F 338
`V 9584
`F. 521
`F 640
`IB4
`F 1219
`F 371
`F 4
`F 6
`F 25
`F 27
`SD4
`F 373
`F 484
`A 33285
`A 35895
`A 10790
`F MG
`F EM48
`F EL28
`F 364
`F Y4
`A 33384
`F 626
`H77
`
`Reactions with
`same species
`
`Reactions with
`heterologous species
`
`Heterologous reactions
`
`Probe results
`
`I/I
`1/1
`I/I
`1/1
`*
`I/I
`"
`3/3
`*
`*
`*
`3/3
`1/1
`1/1
`3/3
`*
`*
`*
`*
`2/2
`1/1
`2/2
`*
`*
`1/1
`1/1
`*
`I/I
`1/1
`1/1
`1/4
`*
`*
`3/4
`2/2
`2/2
`*
`2/2
`
`0/37
`0/37
`1/37
`0/37
`
`0/37
`*
`3/35
`
`0/35
`0/37
`0/37
`3/5
`
`0/36
`2/37
`1/36
`
`0/37
`0/37
`*
`2/37
`0/37
`0/37
`0/34
`
`0/34
`2/36
`2/36
`Hi
`2/36
`
`S. sanguis / C OI
`S. intermedius JS26
`
`W. reeta 371,1219, C. eoneisus 484
`
`W. reeta 371,1219, C. eoneisus 484
`
`C. oehraeea 6, 25
`C. sputigena 4
`
`B. heparinolytieus 35895, B. dentieota 33185
`
`H. aphrophitus H77, 626
`H. aphrophitus Wll, 626
`
`A. aetinomyeetemeomitans Y4, 33384
`
`Total
`A probe was not constructed to this strain. ** F = FDC isolate; A = ATCC isolate; V = VPI isolate.
`
`15/834
`
`36/40
`
`
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`

`fresh hybridization solution (2.5 ml per
`100 cm' of filter) eontaining a denatured
`digoxigenin-labelled probe (the entire
`labelled DNA preparation from 10-20
`colonies). After overnight incubation at
`68°C, filters were washed to remove un-
`bound probe as follows: 2x5 min in
`2 X SSC, 0.1 % (w/v) SDS at room tem-
`perature, then 2x15 min in 0,1 x SSC,
`0.1% (w/v) SDS at 68°C. To detect hy-
`brids, filters were washed briefly in a
`100 mM Tris-HCl, 150 mM NaCl buffer
`(pH 7,5), and incubated at room tem-
`perature with an anti-digoxigenin anti-
`body ~ alkaline phosphatase conjugate,
`diluted l;5000 in the same buffer for 30
`min. Unbound conjugate was removed
`by 2 X 15 min washes in buffer only at
`room temperature. Filters were equilib-
`rated in 100 mM Tris-HCl, 100 mM
`NaCl, 50 mM MgCU (pH 9,5) and incu-
`bated with 10 ml of this buffer contain-
`ing 34 //g/ml nitroblue
`tetrazolium
`(NBT) and 18 fcg/m\ bromo-chloro-in-
`dolyl phosphate (BCIP), Development
`proceeded in the dark for 60 min-24
`h and the reaction was terminated by
`soaking the filters in 10 mM Tris-HCl,
`1 mM EDTA, pH 8.0,
`
`Reverse hybridization
`
`143
`
`Results
`Table 1 lists 38 strains from which DNA
`was purified and fixed to nitrocellulose
`filters. Twenty-three of these strains
`were selected for digoxigenin-labelled
`DNA probe preparation and the results
`of their hybridizations are summarized
`in Table 1, Of 23 probes, 21 detected
`all members of the homologous species
`present on the filters. The 2 exceptions
`were a probe to E ttucteatutn strain MG,
`whieh detected only its homologous
`DNA, and a probe to E ttucteatutn
`strain 364, which detected the 3 remain-
`ing strains of this species. Overall, the
`labelled probes detected the DNAs of
`the same species in 36 of the 40 instances
`when such detection should have oc-
`curred.
`Thirteen of the 23 digoxigenin-labell-
`ed DNA probes showed no false-posi-
`tive reactions with target DNAs from
`heterologous species. For example. Fig.
`1 shows 2, 38-strain nitrocellulose filters
`after hybridization and development.
`The top filter was hybridized with a
`probe to Bacteroides gingivalis 381; a
`single spot indicates the site of probe-
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`1234
`
`1234
`
`Fig. I. Nitrocellulose filters carrying 100 ng targets of purified, denatured DNA from 38
`strains. Row I, left to right: W. reeta 371 and 1219, C eonei.ius 484, B. graeitis 1083 and 406,
`W. .sputigetia IB4, W. eurva 521, 640 and 9584, C. eoneisus 484 (repeat), B. graeitis 402, E.
`eorrodens 373. Row 2: 5. sanguis I COl, S. .'ianguis II Al, S. intetmedius JS26, P. mieros
`JH20, A. naestutidii 12104, blank, F. iiueleatwn 364, MG, EM48 and EL28. Row 3: A.
`aetinomyeetemeomitans Y4, H. aptirophitus VVll and 626, A. aetinomyeetemeomitans 33384,
`blank, V. parvula t0790, C. oehraeea 25, C. sputigena 4, C. gingivalis 27, C. oehraeea 6,
`Capttoeytophaga type IV SD4. Row 4: B. intermedius 25261, 581 and 8944, B.for.sytlms 338,
`B. gingivatis 381, B. dentieota 33185, B. zoogteoformans 33285, B. heparinotytieus 35895, B.
`veroralis 33779. The top filter was hybridized with a digoxigenin-labelled DNA probe prepared
`with B. gingivatis 381 DNA. The lower filter was hybridized with a digoxigenin-labelled DNA
`probe prepared with V. parvuta 10790 DNA. Preparation of probes, target DNAs on filters,
`hybridization conditions and detection of hybrids are described in the text.
`
`calf thymus DNA was added to each
`suspension (10 /d of IOO //g/ml DNA in
`TE), Cell lysis and CTAB extractions
`were performed as described above. Af-
`ter chlorofonniisoamyl extraction, cen-
`trifugation (12,000 xg, 15 min) and re-
`moval of the upper aqueous layers to
`fresh tubes, DNA was precipitated by
`the addition of 0.6 volumes of isopro-
`panol. Suspensions were centrifuged
`(12,000 xg, 5 min) and the precipitates
`washed twice with 70% ethanol. Pre-
`cipitates eolleeted by centrifugation
`(12,000xg, 5 min) were allowed to dry
`in air for approximately 20 min, after
`which 15 /.t\ of a low EDTA-Tris buffer
`(10 mM Tris, 0,1 mM EDTA, pH 7.6)
`was added to each tube. Tubes were
`incubated at 4°C overnight to allow the
`DNA present in precipitates to dissolve
`in the buffer.
`
`Labeiiing of isolated DNA
`A random primer technique (2) was em-
`ployed to construct digoxigenin-labelled
`probes from DNA contained in the
`CTAB preparations, using reagents sup-
`plied by Boehringer Mannheim (Indian-
`apolis, IN). DNA was denatured, by
`heating at 95°C for 10 min, and ineu-
`bated at 37°C for 60 min with 2 //I of a
`random hexanueleotide mixture, 2 /J of
`a dNTP mixture containing dCTP,
`dATP, dGTP (I mmol/l each), dTTP
`(0,65 mmol/l) and digoxigenin-dUTP
`(0,35 mmol/l), sterile distilled water
`upto 19 /(I, and 1 //I Klenow fragment.
`The reaction was stopped by the ad-
`dition of 2 ft\ 0,2 M EDTA, and DNA
`was precipitated by adding 2 /il 4 M
`lithium chloride and 60 /tl ice-cold 95%
`ethanol to the mixtures, which were sub-
`sequently kept at — 20 C for 2 h, or
`— 70"C for 30 min, DNA was collected
`by centrifugation (12,000xg, 2 min),
`washed with ice-cold 70% ethanol,
`dried under vacuum and dissolved in 50
`^1 TE (10 mM Tris, pH 7.6, 1 mM
`EDTA). Hybridization reagents and
`conditions employed for digoxigenin-
`labelled probes were as recommended
`by Boehringer Mannheim. Briefly, fil-
`ters were prehybridized in sealed plastic
`bags for 60 min at 68 C in 20 ml hybrid-
`ization solution per 100 cm- filter. The
`hybridization solution, which consisted
`of 5 X SSC, 1 % (w/v) blocking reagent,
`0.1% (w/v) sodium salt of N-lauroylsar-
`cosine, 0.02% (w/v) SDS, was allowed
`to dissolve at 50-70°C for at least 60
`min prior to use. Following piehybridi-
`zation, this solution was replaced with
`
`
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`

`

`study were speeies-specific. Cross reac-
`tions between closely related strains
`were anticipated, since whole genomic
`DNA was used for probe construction.
`As previously reported (5, 7, 9), heterol-
`ogous probe-target reactions generate
`weaker signals than true-positive reac-
`tions present on the same filters, and
`therefore can be easily distinguished.
`The failure of the 2 E nucteatutn probes
`to detect all members of this speeies
`represented on filters reinforces
`the
`known heterogeneity of this "species"
`(1,4, 6), It is of interest that E imcleatum
`strain MG has been shown to differ
`from the 3 other test strains by SDS-
`PAGE (1), suggesting that DNA probe
`analysis may become a useful method
`to distinguish subgroups within this
`"species".
`The reverse hybridization protocol
`has a number of attraetions. Once pure
`cultures are available on agar media,
`probe construction, hybridizations and
`identifieation of unknown isolates can
`be aecomplished within 2-3 days. Many
`cultures can be processed
`simul-
`taneously. For example, one individual
`can conveniently extract DNA
`for
`probe construction from 48 cultures on
`one day, label the DNA and begin hy-
`bridizations the following day, and de-
`tect the spots the next morning. If
`necessary, labelled DNA preparations
`can be stored at — 20°C for prolonged
`periods of time prior to hybridization.
`Nitrocellulose filters earrying referenee
`strain target DNA ean be prepared in
`bulk and stored until required, either in
`sealed bags at — 20"C, or in a dessieator
`in a cold room. The variety of strains
`represented on filters can be "tailor-ma-
`de", according to the investigator's
`interest, e,g. limited to suspected peri-
`odontopathic species, or expanded to
`encompass dozens or even the full range
`of species commonly found subgingival-
`ly. In terms of time and materials, the
`eost or this identification procedure is
`considerably lower than predominant
`cultivable teehniques.
`Whole chromosomal DNA probes
`have been successfully used to dinstin-
`guish closely related species within the
`genus Bacteroides (5, 8). However, the
`potential diffieulty
`in distinguishing
`closely related species is recognized. If
`the range of eross reactions for each
`referenee strain on a filter is known, it
`should be possible to place unknown
`isolates into groups, which would allow
`their speeific
`identification via ad-
`ditional phenotypic tests, or the use of
`
`144
`
`Smith et al.
`
`target DNA reaction. The lower filter
`was hybridized with a probe to Veil-
`tonetta parvuta 10790; this probe also
`detected DNA from the homologous
`strain only. The remaining 10 probes
`displayed eross reactions with DNA
`from closely related heterologous spe-
`cies present on filters. Labelled DNA
`from Actinobaciltus actinomycetemco-
`mitans strains detected Haemophitus
`aphrophitus and vice versa, while B. gra-
`eilis cross-reacted with Wolinelta recta
`and Campylobacter concisus. As shown
`in Fig, 2, a probe to Bacteroides zoogie-
`oformans cross-reacted with Bacteroides
`heparinotyticus and Bacteroides dentic-
`ota, whereas
`the B. heparinolyticus
`probe was specific. Such false-positive
`reaetions could be distinguished by eye,
`sinee the signals were elearly weaker
`than those from homologous probe-tar-
`get reaetions. In all, cross reaetions oc-
`curred in 15 of 834 possible instances.
`Each target "spot" of DNA on the
`38-strain nitrocellulose filters contained
`100 ng of DNA. In order to determine
`the limits of sensitivity of each probe,
`an additional strip of nitrocellulose was
`ineluded in each hybridization reaction
`carrying 10 ng, 1 ng, 100 pg and 10
`pg spots of the corresponding purified,
`denatured DNA. Development of these
`strips revealed that the digoxigenin-
`labelled probes prepared from 10-20
`bacterial colonies consistently detected
`100 pg of purified homologous DNA.
`
`Discussion
`The purpose of present investigation
`was to determine the feasibility of a "re-
`verse" hybridization method to identify
`pure cultures of subgingival species. In
`this method, DNA was rapidly extract-
`ed from small numbers of bacterial cells
`grown in pure culture, labelled with di-
`goxigenin and hybridized against a
`range of purified, denatured reference
`strain DNAs fixed to nitrocellulose.
`This procedure allowed the identifi-
`cation or grouping of the unknown iso-
`late. The "classical" hybridization tech-
`nique, in which DNA from unknown
`microorganisms is fixed to a solid sup-
`port and probes are eonstructed from
`purified referenee DNAs, was thus re-
`versed. In the present study, DNA was
`labelled with digoxigenin, but previous
`experiments indieated that, using the
`same relatively simple and rapid pro-
`cedure deseribed here, DNA could be
`extracted from 10* cells in a form and
`amount suitable for biotin-labelling
`with the random primer technique, A
`wider range of DNA concentration can
`be incorporated
`in the digoxigenin-
`labelling reaction (10 ng-3 pg, as op-
`posed to 0.1-1 pg DNA in the biotin-
`labelling reaction), and in addition, the
`hybridization protocol recommended
`by Boehringer Mannheim for digoxi-
`genin probes is simpler.
`Many of the probes prepared in this
`
`1234
`
`1234
`
`Fig. 2. Nilrocclliilo.se fillers :is described in Fig. I legend. The top niter was hybridized with
`a digoxigenin-labelled DNA probe prepared with B. heparinotytieus 35895 DNA. The lower
`filter was hybridized with a digoxigenin-labelled DNA probe prepared with B. zoogteoformatis
`33285 DNA. Preparation of probes, target DNAs on filters, hybridization conditions and
`detection of hybrids are described in the text.
`
`
`Exhibit 2016 Page 4
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`

`

`cloned or oligonucleotide probes. Alter-
`natively, it may be possible to avoid
`such cross reactions if species-specific
`cloned DNA fragments or specific
`oligonucleotide sequences were bound
`to filters and the reverse methodology
`employed.
`There are a number of technical ad-
`vantages to the reverse hybridization
`procedure. A major drawback of "clas-
`sical" hybridization procedures em-
`ploying biotin- or digoxigenin-labelled
`probes for strain identification is the
`need
`to remove cellular macromol-
`ecules, such as RNA and protein, from
`filters. Failure in this regard results in
`non-speeifie binding of the deteetion
`complexes used for these probes. Fix-
`ation of purified DNA to nitrocellulose
`for the reverse hybridization procedure
`eliminates the necessity of treating fil-
`ters with enzymes or organic reagents.
`In addition, it appears that relatively
`small amounts of labelled DNA in a
`"sample" probe can find and attach to
`larger amounts of purified target DNA
`on the filter. This suggests that the
`method might be adapted to the identifi-
`cation of a range of species within one
`sample, thus avoiding the problems as-
`sociated with aliquotting samples to
`
`several filters. The procedures described
`here are being investigated further to
`assess their potential for the direct
`identification of suspeeted pathogens
`and beneficial species in subgingival
`plaque samples.
`
`Acknowledgements
`This research was supported in part by
`Grant DE-04881 from The National In-
`stitute for Dental Researeh.
`
`References
`
`1. Dzink JL, Socransky SS, McGillicuddy
`KE, Sheehan MX Amino acid utilization
`patterns of F nueteatum. .1 Dent Res
`1987: 66: 120, Abstr 111.
`2. Feinberg AP, Vogelstein B. A technique
`for radiolabelling DNA restrietion endo-
`nuclease fragments to high speeific ac-
`tivity. Anal Biochem 1983: T32: 6-13.
`3. Grunslein M, Hogness DS. Colony hy-
`bridization: A method for the isolation
`of cloned DNAs that contain a specific
`gene. Proc Natl Aead Sci USA 1975: 72:
`3961-3965.
`4. Lawson PA, Shah HN, Clark DR, De-
`velopment of DNA probes for Fitsobac-
`teria species, J Dent Res 1988: 67: 653,
`Abstr 110.
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`
`145
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`5. Morotomi M, Ohno T, Mutai M. Rapid
`and correct identification of intestinal
`Baeteroides spp. with chromosomal
`DNA probes by whole-cell dot blot hy-
`bridization. Appl Environ Microbiol
`1988: 54: 1158-1162.
`6. Potts TV, Holdeman LV, Slots J. Re-
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`
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`This document is a scanned copy of a printed document. No warranty is given about
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`Exhibit 2016 Page 6
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`Exhibit 2016 Page 6
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