Method for detection, identification and quantitation of non-viral organisms

A method for specifically and sensitively detecting, identifying, and quantitating any non-viral organism, category or group of organisms containing ribosomal RNA in a sample is disclosed. The nucleic acids of the organisms present in the sample are brought together with a marked probe comprising nucleic acid molecules which are complementary only to ribosomal RNA subsequences known to be conserved in an organism, category or group or organisms. The probe and sample nucleic acid mixture is incubated under nucleic acid hybridization conditions and then assayed to determine the degree of hybridization that has occurred. Hybridization indicates the presence and identity of the organism, category or group or organisms in the sample. The quantity of ribosomal RNA present in the sample can be determined and compared to that normally present in the known organisms to determine the number of organisms present. Batteries of sequentially more specific probes can also be utilized.

BACKGROUND OF THE INVENTION 1. Field of the Invention 
The present invention relates to a method and means for detecting, 
identifying, and quantitating non-virus organisms in biological and other 
samples. More particularly the present invention relates to a method for 
specifically and sensitively detecting and quantitating any organism 
containing ribosomal RNA, hereinafter rRNA: any members of large, 
intermediate, or small sized categories or taxonomic groups of such 
organisms; and previously unknown organisms containing rRNA. The method is 
capable of detecting the presence of even one organism containing rRNA. 
My invention has broad application to any area in which it is important to 
detect the presence or absence of living organisms. Such areas include 
medical, veterinary, and agricultural diagnostics, and industrial and 
pharmaceutical biological quality control. 
The invention involves a method of using specifically produced nucleic 
acids complementary to rRNA, hereinafter rRNA probes, to detect, 
quantitate, and identify specific rRNA sequences by a procedure which 
includes nucleic acid hybridization as an essential step. 
My invention and the novelty, utility, and unobviousness thereof can be 
more clearly understood and appreciated when considered in the light of 
the representative background information hereinafter set out, comprising 
this art. 
Each of the cells of all life forms, except viruses, contain ribosomes and 
therefore ribosomal RNA. A ribosome contains three separate single strand 
RNA molecules, namely, a large molecule, a medium sized molecule, and a 
small molecule. The two larger rRNA molecules vary in size in different 
organisms. 
Ribosomal RNA is coded for by the rRNA gene. This DNA sequence is used as a 
template to synthesize rRNA molecules. A separate gene exists for each of 
the ribosomal RNA subunits. Multiple rRNA genes exist in most organisms, 
many higher organisms containing both nuclear and mitochondrial rRNA 
genes. Plants and certain other forms contain nuclear, mitochondrial and 
chloroplast rRNA genes. For simplicity of discussion hereinafter, the 
three separate rRNA genes will be referred to as the rRNA gene. 
Numerous ribosomes are present in all cells of all life forms. About 85-90 
percent of the total in RNA in a typical cell is rRNA. A bacterium such as 
E. coli contains about 10.sup.4 ribosomes per cell while a mammalian liver 
cell contains about 10.times.10.sup.6 ribosomes. Since each ribosome 
contains one of each rRNA subunit, the bacterial cell and mammalian liver 
cell contains 10.sup.4 and 5.times.10.sup.6, respectively, of each rRNA 
subunit. 
Nucleic acid hybridization, a procedure well-known in the art, has been 
used in the prior art to specifically detect extremely small or large 
quantities of a particular nucleic acid sequence, even in the presence of 
a very large excess of non-related sequences. Prior art uses of nucleic 
acid hybridization are found, for example, in publications involving 
molecular genetics of cells and viruses; genetic expression of cells and 
viruses; genetic analysis of life forms; evolution and taxonomy of 
organisms and nucleic acid sequences; molecular mechanisms of disease 
processes; diagnostic methods for specific purposes, including the 
detection of viruses and bacteria in cells and organisms. 
Probably the best characterized and most studied gene and gene product are 
the rRNA gene and rRNA, and the prior art includes use of hybridization of 
rRNA and ribosomal genes in genetic analysis and evolution and taxonomic 
classification of organisms and ribosomal gene sequences. Genetic analysis 
includes, for example, the determination of the numbers of ribosomal RNA 
gene in various organisms; the determination of the similarity between the 
multiple ribosomal RNA genes which are present in cells; determination of 
the rate and extent of synthesis of rRNA in cells and the factors which 
control them. Evolution and taxonomic studies involve comparing the rRNA 
gene base sequence from related and widely different organisms. 
It is known that the ribosomal RNA gene base sequence is at least partially 
similar in widely different organisms, and that the DNA of E. coli 
bacterial ribosomal RNA genes hybridizes well with rRNA from plants, 
mammals, and a wide variety of other bacterial species. The fraction of 
the E. coli gene which hybridizes to these other species varies with the 
degree of relatedness of the organisms. Virtually all of the rRNA gene 
sequence hybridizes to rRNA from closely related bacterial species, while 
less hybridizes to rRNA from distantly related bacterial species, and even 
less with mammalian rRNA. 
2. Description of the Prior Art 
I am not aware of any prior art which teaches my method of detecting the 
presence or absence of rRNA characteristic of a particular group of 
organisms utilizing nucleic acid hybridization wherein is used a selected 
marked nucleic acid molecule complementary to a subsequence of rRNA from a 
particular source. Nor am I aware of any prior art which discloses my 
method for detecting the presence or absence of rRNA in general by nucleic 
acid hybridization using a marked nucleic acid molecule complementary to 
all of the rRNA subsequences from a specific source. 
While the presence of organisms can be detected by any one of a large 
variety of prior art methods, none of these is entirely satisfactory for 
one reason or another. Such methods include, e.g., growth methods, optical 
detection methods, serologic and immunochemical methods, and biochemical 
methods, and are further discussed hereinafter. 
Growth Tests 
A large number of different growth tests exist, each useful for the growth 
of a specific organism or group of organisms. Growth tests have the 
potential sensitivity to detect one organism. In practice, however, many 
organisms are difficult or impossible to grow. These tests are usually 
lengthy, taking from one day, to months, to complete. In addition, a very 
large number of tests would be needed to detect the presence of any member 
of a large group of organisms (e.g., all bacteria), assuming that the 
growth conditions for all members of the group are known. 
Optical Detection Methods 
Microscopic analysis coupled with differential staining methods is a very 
powerful, and in many cases, very rapid detection method. A major problem 
with this approach is the detection of specific organisms in the presence 
of large quantities of other organisms, for example, the identification of 
a specific type of gram negative rod shaped bacteria, in the presence of 
many different kinds of gram negative rod shaped bacteria. In addition, a 
large number of tests would be needed to detect the presence of all 
members of a large group of organisms (such as the group of all bacteria). 
Serologic and Immunochemical Methods and Biochemical Tests 
A large number of different types of these tests exist. They are ususally 
qualitative, not very sensitive and often require a growth step. A great 
many of these tests would be required to detect all members of a large 
group of organisms. 
SUMMARY OF THE INVENTION 
My invention is a simple method and means having, as characterizing 
qualities, (a) the ability to specifically detect the presence of any one 
of a large number of different organisms with a single assay procedure, 
which also works regardless of the pattern of genetic expression of any 
particular organism; (b) the ability to modify the test to detect only 
specific categories of organisms, even in the presence of organisms not in 
the group of interest; (c) extremely high sensitivity of detection, and 
ability to detect the presence of one organism or cell; (d) the ability to 
quantitate the number of organisms or cells present; and (e) does not 
require a growth step. 
As described hereinbefore, rRNA base sequences are partially similar in 
widely different organisms. The more closely related two organisms are, 
the larger the fraction of the total rRNA which is similar in the two 
species. The rRNA sequence of any particular species of organism can be 
regarded as a series of short rRNA subsequences, of which one subsequence 
is similar in virtually all life forms. Therefore, the rRNA of almost all 
life forms must contain this subsequence. A different subsequence is 
similar only in the rRNA of the members of the Species to which that 
organism belongs. Other subsequences are present in the Order of organisms 
that the Species belongs to, and so on. 
Because the rRNA sequences of widely different organisms are at least 
partially similar, the method of my invention, using a probe which detects 
the rRNA sequences which are similar in widely different organisms, can 
detect the presence or absence of any one or more of those organisms in a 
sample. A marked nucleic acid sequence, or sequences complementary to the 
rRNA sequences similar in widely divergent organisms, can be used as such 
a probe in a nucleic acid hybridization assay. 
Because the rRNA sequences of closely related organisms are more similar 
than those of distantly related organisns, the method of my invention, 
which includes using a probe which detects only the rRNA sequences which 
are similar in a particular narrow group of organisms, can detect the 
presence or absence of any one or more of those particular organisms in a 
sample, even in the presence of many non-related organisms. These group 
specific probes can be specific for a variety of different sized 
categories. One probe might be specific for a particular taxonomic Genus, 
while another is specific for a particular Family or another Genus. 
Group specific probes have the ability to hybridize to the rRNA of one 
group of organisms but not hybridize to the rRNA of any other group of 
organisms. Such a group specific complementary sequence will detect the 
presence of rRNA from any member of that specific group of organisms even 
in the presence of a large amount of rRNA from many organisms not 
belonging to that specific group. 
The total number of rRNA molecules in a sample is measured by using a 
marked sequence or sequences complementary to rRNA and standard excess 
probe or excess sample RNA nucleic acid hybridization methodology. 
The rRNA content of cells from a wide variety of organisms is known in the 
art. In a broad group of similar organsims, for example bacteria, the 
amount of rRNA per cell varies roughly 2-5 fold. Therefore, if the number 
of rRNA molecules in a sample, and the broad class identity of the source 
of the rRNA is known, then a good estimate of the number of cells present 
in the sample can be calculated. If the broad class identity is not known 
it can be determined by hybridizing the sample to a series of selected 
probes complementary to rRNA, each of which is specific for a particular 
broad category of organisms. 
At the present time, the operational detection and quantitation range of a 
single assay procedure is from 10.sup.4 rRNA molecules (1 bacterium or 
10.sup.-2 mammalian cells) to about 10.sup.12 rRNA molecules (10.sup.8 
bacteria or 10.sup.6 mammalian cells) a span of about 10.sup.8 in cell 
numbers. A single test could also be done in such a way as to 
operationally quantitate from 10.sup.3 bacteria to 10.sup.10 bacteria. The 
test is quite flexible in this way. 
Because the test for rRNA is specific and has the ability to detect the 
presence of very few organisms there is no need to amplify the numbers of 
organisms through a growth step. 
The practice of that form of my invention which is directed to determining 
the presence of an organism which contains rRNA, in a sample which might 
contain such organism, comprises basically: 
(a) bringing together the sample, or isolated nucleic acids contained in 
that sample, with a probe which comprises marked nucleic acid molecules 
which are complementary to the rRNA of all organisms; 
(b) incubating the resulting mixture under predetermined hybridization 
conditions for a predetermined time, and then; 
(c) assaying the resulting mixture for hybridization of the probe. 
When my invention is directed to determining the presence of any member of 
a specific category of organisms which contain rRNA in a sample which 
might contain such organisms, the method comprises: 
(a) contacting the sample, or the nucleic acids therein, with a probe 
comprising marked nucleic acid molecules which are complementary only to 
the rRNA of members of the specific category of organisms, but not 
complementary to rRNA from non-related organisms; 
(b) incubating the probe and the sample, or the isolated nucleic acids 
therein; and 
(c) assaying the incubated mixture for hybridization of said probe. 
My invention can also be used to determine the number of organisms present 
in the sample under investigation, by adding to the assaying in the second 
above described method in the event probe hybridization has occurred, the 
step of comparing the quantity of rRNA present in the sample with the 
number of rRNA molecules normally present in individual organisms 
belonging to the said specific group. 
And, of course, included in the variations, within the scope of my 
invention, which can be used, is that which comprises, in lieu of the 
single probe of step (a) in the second of the above methods, a 
multiplicity or battery, of different probes. In such case, each separate 
probe comprises marked nucleic acid molecules which are complementary only 
to the rRNA of a specific group of organisms and each probe is specific 
for a different group of organisms; step (a) is followed by incubating 
each probe-sample mixture under predetermined hybridization conditions for 
a pre-determined time, and then assaying each mixture for hybridization of 
the probe. 
PROCEDURES FOR THE PRODUCTION OF GROUP SPECIFIC rRNA PROBES 
Different approaches can be used to produce group specific probes. All of 
these approaches but one, rely on differential nucleic acid hybridization 
methods to identify and purify the group specific probe sequences. 
Procedure A 
The most useful procedure for producing group specific rRNA probes uses 
recombinant DNA methodology. The steps involved in this procedure follow: 
(The specific details of standard DNA recombinant techniques are described 
in the book, Molecular Cloning, A Laboratory Manual, T. Maniatis et al., 
Cold Spring Harbor Publication (1982).) 
1. Isolate nucleic acid from a specific organism of interest. Standard 
isolation methods are used. 
2. Using this isolated DNA, clone the rRNA genes of this organism and then 
produce large amounts of the ribosomal gene DNA, using standard DNA 
recombinant technology, as shown in Maniatis et al., supra. 
3. Reduce the rRNA gene DNA to short pieces with restriction enzymes and 
make a library of these short DNA pieces, using standard DNA recombinant 
methods, as shown in Maniatis et al., supra. 
4. Screen the library and identify a clone which contains a short rRNA gene 
sequence which hybridizes only to rRNA from other members of the taxonomic 
Species of the organism of interest. Isolate this clone. It contains a 
Species specific DNA sequence which is complementary only to the rRNA of 
the specific Species to which the organisms of interest belongs. 
Screen the library further and identify and isolate the following clones: 
(a) a clone which contains a DNA sequence complementary to rRNA which will 
only hybridize to rRNA from members of the taxonomic Genus to which the 
organism of interest belongs; (b) a clone which contains a DNA sequence 
complementary to rRNA which will only hybridize to rRNA from members of 
the taxonomic Order to which the organism of interest belongs; (c) a clone 
which contains a DNA sequence complementary to rRNA which will hybridize 
only to rRNA from members of the taxonomic Family to which the organism of 
interest belongs; (d) a clone which contains a DNA sequence complementary 
to rRNA which will hybridize only to rRNA from members of the taxonomic 
Class to which the organism of interest belongs; and (e) a clone which 
contains a DNA sequence complementary to rRNA which will hybridize to rRNA 
from as many different life forms as possible. 
The foregoing clone selection scheme is only one of a number of possible 
ones. 
Standard methods of cloning and screening are to be utilized, as discussed 
in Maniatis et al., supra. 
5. (a) Produce large amounts of each clone's DNA. From the DNA of each 
individual clone isolate and purify only the DNA sequence which is 
complementary to rRNA, using one of the many methods existing to 
accomplish this, e.g., as in Maniatis et al., supra. 
(b) In certain instances the total DNA present in a clone is useful as a 
probe, in which case the total DNA isolated from the cloning vector is 
used. 
(c) In certain other instances, the DNA single strand of the cloning vector 
which contains the DNA sequence complementary to rRNA is used as a probe. 
In such case this strand must be isolated and purified, using one of the 
various methods which exist to accomplish this, as described by Maniatis 
et al. 
6. The probe DNA obtained in 5a, 5b, and 5c must be marked in some way so 
that it can be identified in the assay mixture. Many different kinds of 
markers can be used, the most frequently used marker being radioactivity. 
Others include fluorescence, enzymes and biotin. Standard methods are used 
for marking the DNA, as set out in Maniatis et al., supra. 
7. The group specific rRNA gene sequence in the cloning vector exists in a 
double strand state. One of these strands is complementary to rRNA and 
will hybridize with it. The other strand will not hybridize to rRNA but 
can be used to produce marked group specific sequences complementary to 
rRNA. This is done by utilizing a DNA or RNA polymerase and nucleic acid 
precursor molecules which are marked. The enzyme will utilize the marked 
precursors for synthesizing DNA or RNA using the DNA strand as a template. 
The newly synthesized marked molecule will be complementary to rRNA and 
can be used as a group specific probe. The template DNA can be removed by 
various established means leaving only single strand marked nucleic acid, 
as described in Maniatis, et al., supra, and the article by Taylor et al., 
in Biochemica and Biophys. Acta (1976) 442, p. 324. 
Procedure B 
Several enzymes can utilize rRNA from any source as a template for the 
synthesizing of marked DNA complementary to the entire rRNA sequence. 
Group specific sequences complementary only to the rRNA of a particular 
class of organisms can be isolated by a hybridization selection process. 
The fraction of the synthesized marked DNA which hybridizes only to the 
rRNA from members of a specific class of organisms can be isolated by 
standard hybridization procedures. An example of this process is presented 
hereinafter. 
Procedure C 
The nucleotide sequences of rRNA from widely different organisms has been 
determined. Group specific sequences similar to a specific group of 
organisms can be identified by comparing these known sequences. A sequence 
complementary to this group specific rRNA sequence can then be chemically 
synthesized and marked, using standard methodology. 
Isolation of Sample Nucleic Acid 
Standard methods are used to isolate the nucleic acid from the samples to 
be assayed; in certain instances nucleic acid hybridization can be done 
without isolating the nucleic acid from the sample. An example of one 
standard method of nucleic acid isolation is presented in the examples and 
also discussed in Maniatis et al., supra. 
The Nucleic Acid Hybridization Test 
Nucleic Acid Hybridization: Two basic methods for performing nucleic acid 
hybridizations are available. In one, in solution hybridization, both the 
probe and sample nucleic acid molecules are free in solution. With the 
other method the sample is immobilized on a solid support and the probe is 
free in solution. Both of these methods are widely used and well 
documented in the literature. An example of the in solution method is 
presented hereinafter in the examples. Also, in the article by Thomas et 
al., Proc. Natl. Acad. Sci. USA (1980), 77 p. 520, is described an 
immobilization method. 
Performing the Nucleic Acid Hybridization 
An appropriate amount of marked probe is mixed with the sample nucleic 
acid. This mixture is then adjusted to a specific salt concentration (NaCl 
is usually used) and the entire mix incubated at a specific temperature 
for a specific time period. At the end of the time period the mixture is 
analyzed by performing a hybridization assay. Many different combinations 
of salt, solvent, nucleic acid concentrations, volumes and temperature 
exist which allow nucleic acid hybridization. The preferred combination 
depends upon the circumstances of the assay. It is important, however, 
that the criteria (see "Definitions") of the hybridization steps be 
identical to criteria used to identify and select the group specific 
probe. If the criteria of the hybridization step are different, the probe 
specificity may change. See: "Repeated Sequences in DNA", by Britten and 
Kohne, Science (1968) 161 p. 529; "Kinetics of Renaturation of DNA", by 
Wetmur and Davidson; J. Mol. Biol. (1968) 31 p. 349; "Hydroxyapatite 
Techniques for Nucleic Acid Reassociation", by Kohne and Britten; 
Procedures in Nucleic Acid Research (1971), eds. Cantoni and Davies, 
Harper and Row, Vol. 2, p. 500. 
Two different approaches are used with regard to the amount of probe and 
sample nucleic acid present in the hybridization mixture. In one, the 
excess probe method, there is more probe present than sample nucleic acid, 
in this case rRNA. With the other, the excess rRNA method, there is more 
rRNA present than probe. The excess probe method is the method of choice 
for detecting the presence of rRNA in unknown samples. It has several 
advantages which are discussed below. See Tables 2 and 3 for further 
discussion of these two approaches. 
Using the excess probe method, the detection and quantitation can be done 
with just one lab assay point, if the proper rRNA probe is available. If 
the hybridization has gone to completion the amount of probe which has 
hybridized is a direct measure of the amount of rRNA present in the 
sample. The fact that the probe hybridizes at all indicates that rRNA is 
present, and the amount of probe which hybridizes indicates the amount of 
rRNA present in the sample. 
Making sure that the hybridization has gone to completion in a known time 
is important in order to quantitate the rRNA. This is readily done by 
adding enough probe to ensure that the hybridization goes to completion in 
a selected time period. The more probe added, the faster completion is 
reached. Thus the excess probe method provides a means to ensure that the 
hybridization has gone to completion and to know when this has occurred. 
In contrast, the detection and quantitation of rRNA can't be done with one 
lab assay point when using the excess rRNA method. In addition, the time 
when the test point should be taken cannot be predicted in the excess rRNA 
method. Unknown samples with small amounts of rRNA will hybridize much 
more slowly than samples with large amounts of rRNA. 
THE ASSAY FOR HYBRIDIZATION 
Quantitation of rRNA 
The signal that rRNA of the specific group is in the sample is the presence 
of double strand marked probe. Many different methods are available for 
assaying the hybridization mixture for the presence of marked probe in the 
double strand form. These methods are well documented in the literature. 
The choice of method depends upon the method chosen for the hybridization 
step, the composition of the hybridization mixture, the type of marker on 
the probe and other factors. One commonly used method is described 
hereinafter. See also Wetmur and Davidson, Kohne and Britten, and Thomas 
et al., supra. Also the article by Flavell et al., Eur. J. Biochem. (1974) 
47 p. 535. And also, the article by Maxwell et al., Nucleic Acids Research 
(1978) 5 p. 2033. 
In all cases, however, it is important to either assay at or above the same 
criterion used for the hybridization reaction or at a criterion at which 
hybridization cannot occur. 
The quantity of rRNA present in a sample can be determined in several ways 
by nucleic acid hybridization, using methods well known to the art. One 
commonly used method is disclosed hereinafter. 
It will be understood that the present method is applicable in any case it 
is necessary to determine the presence or absence of organisms which 
contain rRNA and that such includes biological samples such as sputum, 
serum, tissue swabs, and other animal fluids and tissues as well as 
industrial and pharmaceutical samples and water. 
TABLE 1 
__________________________________________________________________________ 
EXCESS SELECTED PROBE METHOD 
PROBE: The probe is a specific, selected, marked sequence from a 
member of bacteria group B, which 
represents 10 percent of the base sequence of the rRNA, and 
hybridizes completely with rRNA 
from group B bacteria, but does not hybridize with rRNA from 
other organisms. The probe cannot 
hybridize with itself. 
__________________________________________________________________________ 
A. 
Positive Homologous Control 
0.1 micrograms Probe + 
.fwdarw. 
Hybridize to completion and 
.fwdarw. 
(a) 
One percent of the probe will 
form 
assay for double strand probe. 
double strand molecules. 
(b) 
This is a direct measure of 
the rRNA 
10.sup.-3 micrograms Sample group B rRNA in the sample. The number of 
probe 
molecules hybridized equals 
the number 
of rRNA molecules present. 
B. 
Heterologous Control 
0.1 micrograms Probe + 
.fwdarw. 
Hybridize to completion and 
.fwdarw. 
The probe does not hybridize 
with 
assay for double strand probe. 
any rRNA but rRNA from group 
B 
bacteria. 
10.sup.-3 micrograms Sample human rRNA 
C. 
Unknown Sample 
0.1 micrograms Probe + 
.fwdarw. 
Hybridize to completion and 
.fwdarw. 
(a) 
If no group B rRNA is 
present, no 
assay for double strand probe 
probe will hybridize. 
Unknown sample (b) 
If group B rRNA is present, 
the 
probe will hybridize and form 
double 
strand molecules. 
(c) 
The number of probe molecules 
hybridized 
equals the number of group 
.sub.--B rRNA molecules 
present in the sample. 
(d) 
If one percent of the probe 
hybridizes, 
group B rRNA is present since the 
probe 
was selected so that it would 
hybridize 
only with rRNA from group B 
bacteria. 
Since the probe will only 
hybridize to 
group B rRNA, the presence of 
other 
rRNAs will not interfere the 
detection 
or the quantitation of any 
bacterial rRNA 
present 
(e) 
Using a selected probe makes it 
easier to 
ensure that the hybridization is 
complete. 
A selected probe representing 10 
percent 
of the rRNA sequence will 
hybridize 
10 times faster than a probe 
which is 
representative of the total rRNA 
sequence. 
(f) 
The detection of rRNA in general 
is not 
possible since the probe 
hybridizes only 
with group B rRNA. The 
sensitivity of 
detection of group B rRNA is 
extremely 
high. 
D. Summary 
The excess probe method needs just one assay 
point in order to detect and quantitate group 
B organisms. 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
EXCESS rRNA METHOD: THE USE OF A SELECTED PROBE 
PROBE: The probe is a specific, selected, marked sequence from group B 
bacteria, which represents 
one-tenth of the rRNA base sequence of one member of group B. 
The probe hybridizes completely 
with rRNA from group B, but does not hybridize to rRNA from 
other organisms. The probe 
cannot hybridize with itself. 
A. 
Positive Homologous Control 
Sample 0.1 micrograms Group B rRNA + 
.fwdarw. 
Hybridize to completion and 
.fwdarw. 
(a) 
The fraction of probe which 
assay for double strand probe. 
hybridizes is a direct 
measure of 
the similarity between the 
rRNA 
and the probe. In this case 
100 
10.sup.-3 micrograms Probe percent of the probe can 
hybridize. 
(b) 
This percentage is not a 
measure of 
the amount of rRNA present. 
In 
order to determine this the 
kinetics 
of the reaction must be 
determined. 
B. 
Heterologous Control 
Sample 0.1 micrograms human rRNA + 
.fwdarw. 
Hybridize to completion 
.fwdarw. 
The probe does not hybridize 
to 
and assay for double strand 
non-bacterial rRNAs. 
probe 
Probe 10.sup.-3 micrograms 
C. 
Unknown Sample 
Sample + .fwdarw. 
Hybridize to completion 
.fwdarw. 
(a) 
If no group B rRNA is 
present in 
and assay for double strand 
the sample there will be no 
probe. hybridized probe. 
Probe 10.sup.-3 micrograms 
(b) 
If group B rRNA is present 
the 
probe will be hybridized. 
(c) 
The amount of rRNA can't be 
deter- 
mined from the percentage 
hybridi- 
zation at the completion of 
the 
reaction. In order to 
determine 
this the kinetics of the 
hybridi- 
zation must be determined. 
Since 
the probe will hybridize 
with only 
one type of rRNA, the 
kinetic 
determination is simple. 
(d) 
If 100 percent of the probe 
has 
hybridized with the sample, 
this 
means that group B rRNA is 
present 
in the sample. It does not 
indicate 
that only this rRNA is 
present. 
Other rRNAs which do not 
hybridize 
with the probe may also be 
present 
in the sample. 
(e) 
If 100 percent of the probe 
hybridizes 
with the sample, it is 
possible to 
specifically quantitate the 
group 
B rRNA in the presence of 
human 
rRNA by determining the 
kinetics 
of hybridization of the 
probe with 
the sample rRNA. Since the 
probe 
will hybridize only with 
group 
B rRNA such a kinetic 
reaction will 
have only one component, the 
one from 
reacting with group B rRNA. 
(f) 
There are situations where 
the 
hybridization can't go to 
completion. 
In this method the sample 
rRNA must 
drive the hybridization to 
completion, 
since only a very small 
amount of 
probe is present. If there 
is not 
sufficient rRNA in the 
sample, the 
hybridization will not be 
completed. 
The interpretation of such a 
situation 
is discussed below. 
If hybridization of unknown 
sample 
results in 20 percent 
hybridization 
of the probe at the usual 
assay time, 
it is not possible to tell 
if the 
reaction is complete with 
only one 
time-point. It is necessary 
to take 
another point at double the 
original 
time to determine if the 
hybridization 
value increases. If it does 
not 
increase then the 
hybridization is 
complete. In this case the 
rRNA is 
at such low concentration in 
the 
sample that the probe is in 
excess, 
and the number of rRNA 
molecules 
present in the sample is 
equal to the 
number of probe molecules 
hybridized. 
If the hybridization value 
is increased 
the hybridization was not 
over at 
the first time-point. A 
third time- 
point must then be done to 
determine 
whether the reaction was 
over at the 
second time point. 
D. 
Summary 
The excess sample rRNA method needs multiple 
assays points in order to detect and quantitate, 
and is much more time-consuming than the excess 
probe method 
__________________________________________________________________________ 
Use of Selected Probes Complementary to Only a Particular Fraction of the 
rRNA Sequence from a Particular Source to Detect rRNA Versus Use of 
Unselected Probes Complementary to the Entire rRNA Sequence from a 
Particular Source to Detect rRNA 
One aspect of my invention, which comprises using specifically selected 
probes complementary to only a particular fraction of the rRNA sequences 
to detect, quantitate, and identify rRNA has important capabilities and 
advantages over another aspect of the invention, that of using unselected 
probes or sequences complementary to the entire rRNA sequence to detect 
rRNA. The advantages of using a selected probe in both excess rRNA and 
excess probe hybridization methodologies are set forth below. The problems 
with using a completely representative probe are also presented. 
The advantages of using a selected probe over using a completely 
representative rRNA probe, with excess probe hybridization, as well as 
with excess rRNA hybridization, is set out below: 
__________________________________________________________________________ 
Advantages of the Excess Probe Hybridization Method 
Problems with Completely 
Advantages of Using 
Representative rRNA Probe 
Selected Probes 
__________________________________________________________________________ 
1. rRNA can be detected in 
The selected probe can be used to 
a sample with the excess probe 
sensitively and specifically detect 
method but there is no way of 
and quantitate the presence of a 
determining the type of rRNA 
particular rRNA, in an unknown 
present. Thus this probe 
sample when used in an excess probe 
cant't be used to specifically 
hybridization method. This can be 
detect and quantitate the 
done with just one lab assay, even 
presence of a particular 
in the presence of rRNA from other 
rRNA in an unknown sample, 
organisms. 
with the excess probe 
hybridization method. 
2. As stated above, the excess 
The use of a selected probe makes 
probe method cannot be used with 
it possible to use the excess probe 
this probe to detect or quanti- 
method for detecting and quantitating 
tate the presence of a particular 
the presence of a particular rRNA 
rRNA in a sample. For this purpose 
in an unknown sample. This greatly 
the probe must be used in 
simplifies the task. 
the excess rRNA method. 
The excess rRNA method is much 
more time consuming, requires 
much more work, and is much 
more complicated than the excess 
probe method. 
1. rRNA can be detected in 
The selected probe can be used to 
an unknown sample with this 
specifically detect and quantitate 
probe, but in many cases 
the presence of a particular rRNA 
there is no way of determin- 
in an unknown sample in all situations. 
ing the type or quantity of 
This can be done even in the presence 
rRNA which is present. Thus 
of large amount rRNA from other 
in many instances the probe 
organisms. 
cannot be used to specific- 
ally detect and quantitate 
the presence of a particular 
rRNA in an unknown sample 
2. In many cases the sensitivity 
With the selected probe the presence 
of detection of a specific rRNA 
of rRNA from other organisms does 
is limited by the presence of 
not lower the sensitivity of detection 
rRNA from other organisms. 
of a particular rRNA. 
3. In many cases where it is 
The detection and quantitation of the 
possible to detect and quanti- 
presence of a particular rRNA is much 
tate the presence of particu- 
easier when a selected probe is utilized 
lar rRNA, it requires a lot 
of work 
__________________________________________________________________________ 
ILLUSTRATIVE EMBODIMENT 
My invention, illustratively, may be used to determine whether a tissue 
culture cell line, or a human or other mammalian tissue, is contaminated 
with any bacteria-like organisms. 
In a typical situation, about 10.sup.6 -10.sup.7 mammalian cells are grown 
in a tissue culture plate at one time. Bacterial species, especially 
members of the taxonomic Class Mollicutes, are known to contaminate tissue 
culture cells. Members of the Class Mollicutes, unlike most other 
bacteria, are not readily eliminated by antibiotics, and are troublesome 
contaminants of cell cultures. Many different Mollicutes species have been 
detected in tissue culture cells. If just one of these organisms is 
present in the culture plate, it has the potential, even in the presence 
of antibiotics, to multiply and produce hundreds of organisms per cell. 
Such organisms are capable of altering the activity of cells, thereby 
affecting the results of various studies, and the marketability of cell 
culture products. 
Prior art methods for detecting these organisms involve basically 
qualitative tests, the most commonly used being growth tests, differential 
staining tests and immunologic assays. The growth tests, while quite 
sensitive, take 3-6 weeks. They have the additional disadvantage that many 
organisms are difficult or impossible to grow. 
While the actual detection sensitivity of the staining method is not known, 
it is known that more than several organisms per cell have to be present. 
Immunologic tests are qualitative tests and involve using antibody toward a 
particular species. While they can be carried out rapidly, they are not 
very sensitive; furthermore, many different antibodies would be required 
to detect all types of Mollicutes. 
The embodiment of applicant's method described in Example I, below, is a 
test which may be used to detect and quantitate the presence of any member 
of the group of all bacteria, including the taxonomic Class Mollicutes, to 
detect the presence of Mollicutes in tissue culture, to detect the 
presence of bacteria in tissue which is normally free of bacteria, and to 
detect the presence of the bacteria even in the presence of large numbers 
of mammalian cells. 
As set forth in the example, applicant's method involves first making a 
specific rRNA probe which is complementary to rRNA from any bacteria but 
is not complementary to mammalian cell rRNA. The use of such a probe in a 
nucleic acid hybridization test allows the detection of any bacteria type, 
even in the presence of large numbers of mammalian cells.

A detailed description of this embodiment of the invention follows: 
EXAMPLE I 
Preparation of rRNA from Mammalian and Bacterial Cells 
Mammalian cells are resuspended in 0.3 M NaCl, 0.02 M Tris, pH=7.4. 
Sarkosyl is added to a final concentration of 1 percent to lyse the cells. 
Immediately upon lysis an equal volume of a 1/1 mixture of 
phenol/chloroform is added and the resulting mixture shaken vigorously for 
2 minutes. The mixture is then centrifuged (8000 x for 10 minutes) to 
separate the aqueous and organic phases. The aqueous phase is recovered, 
and to this is added another volume of phenol/chloroform. After shaking 
and centrifugation as above, the aqueous phase is again recovered. To this 
is added 2 volumes of 95% ethanol and this mixture is placed at 
-20.degree. C. for 2 hours to facilitate precipitation of the nucleic 
acids. Then the mixture is centrifuged (8000 x g, 10 minutes) in order to 
sediment the precipitate to the bottom of the tube. The liquid is then 
removed. The pelleted nucleic acid is redissolved in water. This solution 
is then made to 0.2 M NaCl, 5.times.10.sup. -3 M MgCl.sub.2, 
5.times.10.sup.-3 M CaCl.sub.2. 0.02 M Tris (pH=7.4), 50 micrograms per ml 
of deoxyribonuclease I and incubated at 37.degree. C. for 1 hour. Then add 
an equal volume of phenol/chloroform and shake as above. Centrifuge as 
above and recover the aqueous phase. Ethanol precipate the RNA as above. 
Centrifuge the precipitate as above and redissolve the pelleted RNA in 
water. Make this solution to 2 M LiCl and place it at 4.degree. C. for 
10-20 hours in order to facilitate the precipitation of the high molecular 
weight RNA. Then centrifuge this solution to collect the precipate and 
redissolve the precipitate in water. This preparation of RNA contains 
greater than 95% rRNA. 
Bacterial rRNA is isolated in a similar manner with the following 
exceptions. In those cases where detergent alone does not lyse the 
bacteria, other means are employed. This usually involves pretreating the 
bacteria with an enzyme (lysozyme) to make them susceptible to lysis by 
sarkosyl. After lysis of the bacteria the isolation procedure is as 
described above. 
Purified rRNA is stored at -70.degree. C. 
Production of Radioactive DNA Complementary (.sup.3 H-cDNA) to Mollicutes 
rRNA 
rRNA from the species Mycoplasma hominis (M. hominis), a member of the 
taxonomic class Mollicutes, is used as a template to synthesize 
radioactive cDNA complementary to M. hominis rRNA. 
This cDNA is produced by utilizing the ability of an enzyme, reverse 
transcriptase, to utilize rRNA as a template and produce .sup.3 H-cDNA 
complementary (cDNA) to rRNA. The reverse transcriptate reaction mixture 
contains the following: 50 mM Tris.multidot.HCl (pH=8.3), 8 mM MgCl.sub.2, 
0.4 mM dithiothreitol, 50 mM KCl, 0.1 mM .sup.3 
H-deoxythymidinetriphosphate (50 curies per millimole), 0.2 mM 
deoxyadenosintriphosphate, 0.2 mM deoxycytidinetriphosphate, 0.2 mM 
deoxyguanosinetriphosphate, 200 micrograms per ml of 
oligodeoxyribonucleotide primer made from E. coli DNA, 50 micrograms per 
ml of M. hominis rRNA and 50 units per ml of AMV reverse transcriptase. 
This mixture is incubated at 40.degree. C. for 30 minutes. Then ethylene 
diamine tetraacetic acid (EDTA) (pH=7.3), sodium dodecyl sulfate (SDS), 
NaCl and glycogen are added to final concentrations of 10.sup.-2 M, 1 
percent, 0.3 M, and 100 micrograms per ml respectively. The solution is 
then mixed with 1 volume of phenol/chloroform (1/1) and shaken vigorously 
for 2 minutes, then centrifuged (8000 x g for 10 minutes) and the aqueous 
phase recovered. The nucleic acids are precipitated by the addition of 2.5 
volumes of 95% ethanol. The precipitate is recovered by centrifugation and 
redissolved in H.sub.2 O. This solution contains the template rRNA and the 
newly synthesized .sup.3 H-cDNA. 
This solution is then, made to 0.3 M NaOH and incubated at 50.degree. C. 
for 45 minutes, and cooled in ice and neutralized with 0.3 M HCl. Two and 
one-half volumes of 95% EtOH are then added to precipitate the remaining 
nucleic acid and the resulting precipitate redissolved in water. This 
solution is then passed over a Sephadex G-100 column equilibrated to 0.3 M 
NaCl, 0.1 percent sarkosyl and the excluded volume recovered. This 
solution is ethanol precipitated and the resulting precipitate redissolved 
in a small volume of water. The process described in this paragraph 
removes the template rRNA and any remaining precursor material from the 
.sup.3 H-cDNA preparation. 
The .sup.3 H-cDNA is then hybridized to M. hominis rRNA to ensure that it 
is indeed complementary to this rRNA. The hybridization mixture consists 
of, 0.05 micrograms of single strand .sup.3 H-cDNA, 20 micrograms of M. 
hominis rRNA, and 0.48 M PB (phosphate buffer) in 1 ml. This mixture is 
incubated for 0.2 hours at 65.degree. C. and is then diluted to 0.14 M PB 
and passed over a hydroxyapatite (HA) column equilibrated to 0.14 M PB and 
65.degree. C. .sup.3 H-cDNA hybridized to rRNA adsorbs to the 
hydroxyapatite (HA) column while non-hybridized .sup.3 H-cDNA passes 
through the column. The hybridized .sup.3 H-cDNA is then recovered by 
elution of the HA column with 0.3 M PB. The fraction is then dialysed to 
remove the PB, ethanol precipitated to concentrate the nucleic acid, 
centrifuged and the nucleic acid redissolved in water. The solution is 
then treated with NaOH as described above in order to remove the rRNA. 
After neutralization, addition of glycogen carrier and concentration by 
ethanol precipitation, the .sup.3 H-cDNA is redissolved in a small volume 
of water. This solution contains only .sup.3 H-cDNA which is complementary 
to M. hominis rRNA. 
Selection of .sup.3 H-cDNA Which is Complementary to M. hominis rRNA but is 
not Complementary to Human rRNA 
The purified .sup.3 H-cDNA is further fractionated by hybridizing it with a 
great excess of human rRNA. The hybridization mixture consists of 0.05 
micrograms .sup.3 H-cDNA, and 40 micrograms of human rRNA in one ml of 
0.48 M PB. This is incubated at 68.degree. C. for 1 hour and the mixture 
is then diluted to 0.14 M PB and passed over HA equilibrated to 55.degree. 
C. and 0.14 M PB. The fraction (about 50% of the total) which does not 
adsorb to the HA (i.e., .sup.3 H-cDNA not hybridized to human rRNA) is 
collected. This fraction is repassed over a new HA column under the same 
conditions. Again the non-adsorbed fraction is collected. This fraction is 
dialysed to remove the PB, ethanol precipitated to concentrate the nucleic 
acid and redissolved in water. This solution is treated with NaOH, as 
described earlier, in order to remove the human rRNA. After 
neutralization, addition of glycogen carrier, and concentration by ethanol 
precipitation, the .sup.3 H-cDNA is redissolved in a small volume of 
water. This .sup.3 H-cDNA preparation is complementary to M. hominis rRNA 
but is not complementary to human rRNA. 
Hybridization of Selected .sup.3 H-cDNA with rRNA from Different Sources 
The production of the selected .sup.3 H-cDNA probe allows the detection of 
bacteria, including members of the Class Mollicutes in mammalian tissue 
culture cells and mammalian tissues by detecting the presence of bacterial 
rRNA by nucleic acid hybridization. A necessary requirement of such a test 
is that the selected probe must not hybridize to rRNA from mammalian cells 
which do not contain bacteria. That this requirement is met is shown in 
Table 4V. 
Table 4, parts II and III show that the probe will detect all members of 
the class Mollicutes and should detect all types of bacteria. For example, 
Legionella p. and E. coli and Bacillus subtilis are representatives of 
very different bacterial types and the probe hybridizes with rRNA from 
each of these types. Evolutionary considerations indicate that this probe 
will hybridize to rRNA from virtually any known or unknown bacteria. This 
is due to the extreme conservation of the rRNA nucleotide sequences during 
evolution. 
This selected probe is useful for detecting the presence of a specific 
Class of bacteria, Mollicutes, in tissue culture cells. In most tissue 
culture cells antibiotics are present in the growth medium and this 
prevents the growth of virtually all bacteria but members of the Class 
Mollicutes. Thus any contamination of a tissue culture preparation is 
almost certain to be due to a member of the Class Mollicutes. 
An important aspect is the ability to determine the number of organisms 
present. In most cases, cell lines and their products are discarded when 
cells are shown, by prior art methods, to be contaminated. The ability to 
quantitate these organisms makes it possible to make judgements as to the 
severity of any effects due to contamination. The degree of a 
contamination may be very light, and only one organism per 1000 cells 
present. This level of contamination would have very little effect on the 
cells and in many situations the cell products need not be discarded. The 
decision might be made to retain valuable cell lines until they become 
more heavily contaminated. Quantatitive considerations are important for 
judging the importance of any kind of a bacterial contamination. 
TABLE 4 
__________________________________________________________________________ 
Hybridization of Selected Mollicutes .sup.3 H--cDNA 
with rRNA from Widely Different Sources 
Percent Hybridization 
Source of rRNA of .sup.3 H--cDNA with 
__________________________________________________________________________ 
rRNA 
I. Control A. 
No rRNA added, 
Experiments 
Self Reaction of .sup.3 H--cDNA 
&lt;1% 
B. 
Mock rRNA isolation &lt;1% 
C. 
Human cell RNA known to be contamin- 
ated M. hominis rRNA 97% 
II. 
Hybridization 
A. 
Members of the Order Mycoplasmatales 
of .sup.3 H--cDNA with 
1. Mycoplasma hominis (infects humans) 
97% 
rRNA from dif- 
2. Mycoplasma salivarium (infects humans) 
93% 
ferent species 
3. Mycoplasma hyorhinis (infects pigs) 
84% 
of the taxon- 
4. Mycoplasma pulmonis (infects mice) 
82% 
omic Class 
Mollicutes 
B. 
Members of the Order Acholeplasmataceae 
1. Acholeplasma laidlawii isolate #1 
52% 
(infects cows, birds, dogs, house cats, 
mice, sheep, pigs and primates) 
2. Acholeplasma laidlawii isolate #2 
53% 
II. C. 
Members of the Order Spiroplasmataceae 
1. SMCA (infects insects and mice) 
69% 
2. Honey bee (isolated from honey bee) 
68% 
3. Cactus (isolated from cactus) 
71% 
4. Corn Stunt (isolated from corn) 
69% 
5. Corn Stunt (isolated from insect) 
65% 
III. 
Hybridization 
A. 
Member of the Family Enterobacteraceae 
of .sup.3 H--cDNA with 
1. Escherischia coli (infects mammals) 
52% 
rRNA from other 
types of bacteria 
B. 
Member of the Family Legionellaceae 
(taxonomic Class 
1. Legionella pneumophila (infects man) 
&gt;28% 
Schizomytes) 
C. 
Member of the Family Micrococcaceae 
1. Micrococcus luteus 
50-60% 
2. Staphylococcus aureus 
&gt;50% 
D. 
Member of the Family Lactobacillaceae 
1. Streptococcus faecalis 
&gt;50% 
E. 
Member of the Family Bacillaceae 
1. Bacillus subtilis &gt;40% 
IV. 
Hybridization 
of .sup.3 H--cDNA with &gt;2% 
rRNA from a 
Yeast 
V. Hybridization 
Human (primate) &lt;1% 
of .sup.3 H--cDNA with 
Cow (bovine) &lt;1% 
rRNA from mam- 
mals and a bird. 
Mouse (rodent) &lt;1% 
Rat (rodent) &lt;1% 
Hamster (rodent) &lt;1% 
Rabbit (lagomorph) &lt;1% 
Chicken (avian) &lt;1% 
__________________________________________________________________________ 
Excess rRNA hybridizations are done at 68.degree. C., 0.48 M PB. 
Hybridization assays are done with hydroxyapatite at 67.degree. C. in 0.1 
M PB, 0.005% sodium dodecyl sulfate. The hybridization exposrue is 
sufficient to ensure complete reaction of the .sup.3 H--cDNA with nuclear 
rRNA or for mitochondrial rRNA. Nonbacterial rRNA Cot's of at least 2 
.times. 10.sup.3 are reached in the case of the mammals and bird. A 
nonspecific signal of 1-2 percent has been subtracted from the 
hybridization values presented above. 
TABLE 4, continued. 
Excess rRNA hybridizations are done at 68.degree. C., 0.48 M PB.: 
Hybridization assays are done with hydroxyapatite at 67.degree. C. in 0.14 
M PB, 0.005% sodium dodecyl sulfate. The hybridization exposure is 
sufficient to ensure complete reaction of the .sup.3 H-cDNA with nuclear 
rRNA or for mitochondrial rRNA, Nonbacterial rRNA Cot's of at least 
2.times.10.sup.3 are reached in the case of the mammals and bird. A 
non-specific signal of 1-2 percent has been subtracted from the 
hybridization values presented above. 
Quantitation of rRNA by Nucleic Acid Hybridization 
The amount of bacterial rRNA present in a sample can be determined by 
measuring the kinetics of hybridization of the selected .sup.3 H-cDNA 
probe with the RNA isolated from a tissue sample and comparing these 
kinetics to those of a known standard mixture. This can be done even in 
the presence of a large excess of mammalian cell rRNA since the probe does 
not hybridize with this rRNA (see Table 4,V). 
For measuring the kinetics, the hybridization mixtures contain, 10.sup.-5 
to 10.sup.-4 micrograms of .sup.3 H-cDNA and 1 to 10.sup.3 micrograms of 
purified sample RNA in 0.01 to 0.1 ml of 0.48 M PB. This mixture is 
incubated at 68.degree. C. and aliquots are removed, diluted to 0.14 M PB 
and assayed for hybridization at various times after the initiation of the 
reaction. Hybridization assays are performed using hydroxyapatite as 
described earlier. The results obtained are compared to the hybridization 
kinetics of the probe reacted with standard RNAs containing known amounts 
of bacterial rRNA. These standards are mixtures of mammalian cell RNA and 
known amounts of a specific bacterial rRNA. 
Detection and Quantitation of Members of the Class Mollicutes in Tissue 
Culture Cells 
Table 5 presents data obtained by hybridizing the selected probe with RNA 
isolated (as described earlier) from three different tissue culture cell 
samples. Only cell line number 3 is detectably contaminated and the 
kinetics of the reaction indicate that about 5.times.10.sup.7 bacterial 
cells are present in the tissue culture cells. 
TABLE 5 
__________________________________________________________________________ 
Detection and Quantitation of Mollicutes in Tissue Culture Cells 
Hybridization 
Percent Hybridization 
Number of Bacteria 
Cell Line Time (hours) 
of .sup.3 H--cDNA with RNA 
Detected 
__________________________________________________________________________ 
1. 44-2C (rat) 
17 &lt;1 None detected 
40 &lt;1 None detected 
2. P388 D1M (mouse) 
1.1 &lt;1 None detected 
22.5 &lt;1 None detected 
3. P388 D1C (mouse) 
0.025 20 5 .times. 10.sup.7 
16.2 78 (about 1 Mollicute 
per mammalian cell 
__________________________________________________________________________ 
Excess rRNA Hybridizations are done at 68.degree. C. in 0.48 M PB in a 
volume of 0.01 to 0.04 ml. Each mixture contains 2 .times. 10.sup.5 
micrograms of .sup.3 H--cDNA probe and 50-200 micrograms of sample RNA. 
The following example is another embodiment of the method of my invention, 
used for detecting very small numbers, even one trypanosome, in the 
presence of a large number of blood cells. 
The detection of trypanosomes is important since certain members of the 
protozoan group Trypanosoma are pathogenic for humans, causing diseases 
that include East African sleeping sickness, West African sleeping 
sickness, and South American trypanosomiasis. These organisms are large 
and have varying characteristic shapes, depending on the stage of the life 
cycle. Prior art methods rely mainly on serologic, differential staining 
coupled with microscopic examination and animal inoculation procedures for 
detecting these organisms in humans. The serodiagnostic methods vary in 
sensitivity and specificity and may be difficult to interpret. The 
microscopic methods are most used, however small numbers of the 
trypanosomes are often difficult to detect in the presence of large 
numbers of blood cells. Animal inoculation is a long and costly procedure. 
The embodiment of the invention set forth in the example following is a 
method which makes it relatively easy to detect the presence of one 
trypanosome even when co-present with a large number of blood cells. 
EXAMPLE II 
Production of Radioactive DNA Complementary to Trypanosome rRNA 
Radioactive DNA complementary (.sup.3 H-cDNA) to Trypanosoma brucei rRNA is 
produced in the same way as M. hominis .sup.3 H-cDNA, which is described 
above in detail, except that Trypanosoma b. rRNA is used as a template. 
Selection of Trypanosome .sup.3 H-cDNA Which is Complementary to 
Trypanosome rRNA but is not Complementary to Human rRNA 
This is done in the same way as described earlier for M. hominis except 
that Trypanosoma b. .sup.3 H-cDNA is hybridized to the human rRNA. 
Use of Selected Trypanosome .sup.3 H-cDNA to Detect and Quantitate 
Trypanosomes in Human Tissue or Fluid 
The production of the selected .sup.3 H-cDNA probe allows the detection and 
quantitation of trypanosomes in human samples by detecting the presence of 
trypanosome rRNA. A necessary requirement of such a test is that the 
selected probe must not hybridize to rRNA from human cells which do not 
contain trypanosomes. Table 7 shows that this requirement is met. 
TABLE 7 
______________________________________ 
Hybridization of Selected Trypanosoma brucei 
.sup.3 H--cDNA with rRNA from Different Sources 
Percent Hybridization 
rRNA Source of .sup.3 H--cDNA with rRNA 
______________________________________ 
No RNA added &lt;1% 
Trypanosome brucei rRNA 
98% 
Bacterial (Mycoplasma hominis) rRNA 
&lt;1% 
Human rRNA &lt;1% 
Human rRNA known to be contaminated 
with Trypanosome brucei 
______________________________________ 
Excess rRNA hybridizations are done at 65.degree. C. in 0.48 M PB. 
Reactions are run for 24 hours and the hybridization exposure is 
sufficient to ensure complete reaction of the human nuclear or 
mitochondrial rRNAs and the bacterial rRNA. Hybridization assays are done 
with hydroxyapatite at 72.degree. C. in 0.14 M PB, 0.005% sodium dodecyl 
sulfate. 
The publications listed below are of interest in connection with various 
aspects of the invention and are incorporated herein as part of the 
disclosure. 
1. Repeated Sequences in DNA. R. J. Britten and D. E. Kohne, Science (1968) 
161 p 529. 
2. Kinetics of Renaturation of DNA. J. G. Wetmur and N. Davidson, J. Mol. 
Biol. (1968) 31 p 349. 
3. Hydroxyapatite Techniques for Nucleic Acid Reassociation. D. E. Kohne 
and R. J. Britten, in Procedures in Nucleic Acid Research (1971). eds 
Cantoni and Davies, Harper and Row Vol 2, p 500. 
4. Hybridization of Denatured RNA and Small DNA Fragments Transferred to 
Nitrocellulose. P. S. Thomas, Proc. Natl. Acad. Sci. USA (1980) 77 p 5201. 
5. DNA-DNA Hybridization on Nitrocellulose Filters: General Considerations 
and Non-Ideal Kinetics. R. Flavell el al., Eur. J. Biochem. (1974) 47 p 
535. 
6. Assay of DNA-RNA Hybrids by S.sub.1 Nuclease Digestion and Adsorption to 
DEAE-Cellulose Filters. I. Maxwell et al., Nucleic Acids Research (1978) 5 
p 2033. 
7. Molecular Cloning: A Laboratory Manual. T. Maniatis et al., Cold Spring 
Harbor Publication (1982). 
8. Efficient Transcription of RNA into DNA by Avian Sarcoma Virus 
Polymerase. J. Taylor et al., Biochemica et Biophys. Acta (1976) 442 p 
324. 
9. Use of Specific Radioactive Probes to Study Transcription and 
Replication of the Influenza Virus Genome. J. Taylor et al., J. Virology 
(1977) 21 #2, p 530. 
10. Virus Detection by Nucleic Acid Hybridization: Examination of Normal 
and ALs Tissue for the Presence of Poliovirus. D. Kohne et al., Journal of 
General Virology (1981) 56 p 223-233. 
11. Leukemogenesis by Bovine Leukemia Virus. R. Kettmann et al., Proc. 
Natl. Acad. Sci. USA (1982) 79 #8 p 2465-2469. 
12. Prenatal Diagnosis of .alpha. Thalassemia: Clinical Application of 
Molecular Hybridization. Y. Kan et al., New England Journal of Medicine 
(1976) 295 #21 p 1165-1167. 
13. Gene Deletions in .alpha. Thalassemia Prove that the 5' Locus is 
Functional. L. Pressley et al., Proc. Natl. Acad. Sci. USA (1980) 77 #6 p 
3586-3589. 
14. Use of Synthetic Oligonucleotides as Hybridization Probes. S. V. Suggs 
et al., Proc. Natl. Acad. Sci. USA (1981) 78 p 6613. 
15. Identification of Enterotoxigenic E. coli by Colony Hybridization Using 
3 Enterotoxin Gene Probes. S. L. Mosely et al., J. of Infect. Diseases 
(1982) 145 #6 p 863. 
16. DNA Reassociation in the Taxonomy of Enteric Bacteria D. Brenner, Int. 
J. Systematic Bacteriology (1973) 23 #4 p 298-307. 
17. Comparative Study Ribosomal RNA Cistrons in Enterobacteria and 
Myxobacteria. R. Moore et al., J. Bacteriology (1967) 94 p 1066-1074. 
18. Ribosomal RNA Similarities in the Classification of Rhodococcus and 
Related Taxa. M. Mordarski et al., J. General Microbiology (1980) 118 p 
313-319. 
19. Retention of Common Nucleotide Sequences in the Ribosomal RNA DNA of 
Eukaryotes and Some of their Physical Characteristics. J. Sinclair et al., 
Biochemistry (1971) 10 p 2761. 
20. Homologies Among Ribosomal RNA and Messenger RNA Genes in Chloroplasts, 
Mitochondria and E. coli. H. Bohnert et al., Molecular and General 
Genetics (1980) 179 p 539-545. 
21. Heterogeneity of the Conserved Ribosomal RNA Sequences of Bacillus 
subtilis. R. Doi et al., J. Bacteriology (1966) 92 #1 p 88. 
22. Isolation and Characterization of Bacterial Ribosoma RNA Cistrons. D. 
Kohne, Biophysical Journal (1968) 8 #10 p 1104-1118. 
23. Taxonomic Relations Between Archaebacteria Including 6 Novel Genera 
Examined by Cross Hybridization of DNAs and 16S rRNAs. J. Tu et al., J. 
Mol. Evol. (1982) 18 p 109. 
24. rRNA Cistron Homologies Among Hyphomicrobium and Various Other 
Bacteria. R. Moore, Canadian J. Microbiology (1977) 23 p 478. 
As used in the specification and claims the following terms are defined as 
follows: 
__________________________________________________________________________ 
DEFINITION OF TERMS 
__________________________________________________________________________ 
base (see nucleotide) 
base pair mismatches 
(see imperfectly complementary 
base sequence) 
base sequence, (nucleotide 
sequence or gene sequence 
or polynucleotide sequence 
A DNA or RNA molecule consisting of 
or single strand nucleic 
multiple bases. 
acid sequence) 
complementary base pairs 
Certain of the bases have a chemical 
affinity for each other and pair together 
or are complementary to one another. 
The complementary base pairs are A:T 
and G:C in DNA and A:U and G:C in RNA 
complementary strands of 
Perfectly complementary nucleic acid 
complementary base sequences 
molecules are nucleic acids molecules 
in which each base in one molecule 
is paired with its complementary base 
in the other strand, to form a stable 
helical double strand molecule. The 
individual strands are tempted complementary 
strands 
criterion Most precisely defined as the difference 
between the temperature of melting of 
the double strand nucleic acid and the 
temperature at which hybridization is 
done. The melting temperature of a 
double strand nucleic acid is determined 
primarily by the salt concentration of 
the solution. The criterion determines 
the degree of complementarity needed for 
two single strands to form a stable double 
strand molecule. The criterion can be 
described as highly stringent, stringent, 
or not very stringent. A highly stringent 
criterion requires that two interacting 
complementary sequences be highly comple- 
mentary in sequence in order to form a 
stable double strand molecule. A 
poorly stringent criterion is one 
which allows relatively dissimilar 
complementary strands to interact 
and form a double strand molecule. 
High stringency allows the presence 
of only a small fraction of base pair 
mismatches in a double strand molecule 
A poorly stringent criterion allows a 
much larger fraction of base pair 
mismatches in the hybridization product. 
denatured or dissociated 
The bonds between the paired bases 
nucleic acid in a double strand nucleic acid 
molecule can be broken, resulting 
in two single strand molecules, which 
then diffuse away from each other. 
double strand nucleic acid 
As it is found in the cell, most 
DNA is in the double strand state. 
The DNA is made up of two DNA molecules 
or strands wound helically around each 
other. The bases face inward and each 
base is specifically bonded to a comple- 
mentary base in the other strand. For 
example, an A in one strand is always 
paired with a T in the other strand, 
while a G in one strand is paired with 
a C in the other strand. In a bacterial 
cell the double strand molecule is about 
5 .times. 10.sup.6 base pairs long. Each of the 
bases in one strand of this molecule 
is paired with its base complement in 
the other strand. The base sequences 
of the individual double strand molecules 
are termed complementary strands. 
hybridization 
(see nucleic acid hybridization) 
imperfectly complementary 
Stable double strand molecules can 
base sequences (base pair 
be formed between two strands where 
mismatches) a fraction of the bases in the 
one strand are paired with a non-comple- 
mentary base in the other strand. 
marked probe or Single strand nucleic acid molecules 
marked sequence which are used to detect the presence 
of other nucleic acids by the process 
of nucleic acid hybridization. The 
probe molecules are marked so that 
they can be specifically detected. 
This is done by incorporating a specific 
marker molecule into the nucleic acid 
or by attaching a specific marker to 
the nucleic acid. The most effective 
probes are marked, single strand 
sequences, which cannot self hybridize 
but can hybridize only if the nucleic 
acid to be detected is present. 
A large number of different markers are 
available. These include radioactive 
and fluorescent molecules. 
nucleic acid hybridization 
The bonds between the two strands of 
or hybridization, (reassociation, 
a double strand molecule can be broken 
or renaturation) 
and the two single strands can be 
completely separated from each other. 
Under the proper conditions the 
complementary single strands can 
collide, recognize each other and 
reform the double strand helical 
molecule. This process of formation 
of double strand molecules from 
complementary single strand molecules 
is called nucleic acid hybridization. 
Nucleic acid hybridization also occurs 
between partially complementary single 
strands 
nucleotide, nucleotide base 
Most DNA consists of sequences of only 
or base four nitrogeneous bases: adenine (A), 
thymine (T), guanine (G), and cytosine 
(C). Together these bases form the 
genetic alphabet, and long ordered 
sequences of them contain, in coded 
form, much of the information present 
in genes. 
Most RNA also consists of sequences of 
only four bases. However, in RNA, 
thymine is replaced by uridine (U). 
reassociation (see nucleic acid hybridization) 
renaturation (see nucleic acid hybridization) 
ribosomal RNA or rRNA 
The RNA which is present in ribosomes. 
Virtually all ribosomes contain 3 
single strand RNA subunits: one large, 
one medium sized, and one small. 
ribosome A cellular particle (containing RNA and 
protein) necessary for protein synthesis. 
All life forms except viruses contain 
ribosomes. 
rRNA DNA or The base sequence in the DNA which 
rRNA gene codes for ribosomal RNA. Each rRNA 
subunit is coded for by a separate 
gene. 
rRNA probe A marked nucleic acid sequence which is 
complementary to rRNA and therefore 
will hybridize with rRNA to form a 
stable double strand molecule. 
thermal stability of 
The thermal stability or melting 
double strand nucleic 
temperature is the temperature at 
acid molecules which half of a population of double 
strand molecules has been converted 
to the single strand form. 
restriction enzymes 
Components of the restriction-modification 
cellular defense system against foreign 
nucleic acids. These enzymes cut 
unmodified (e.g., methylated) double- 
stranded DNA at specific sequences which 
exhibit twofold symmetry about a point. 
__________________________________________________________________________ 
While the invention has been described in detail for purposes of 
illustration, and to meet the requirements of 35 USC 112, it will be 
apparent to those skilled in the art that changes and modifications 
therein are included without departing from the spirit and scope of the 
invention.