JP3178014B2 - Method for measuring RNA - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to nucleic acid diagnosis which has attracted attention in the field of clinical diagnosis, and more particularly to measurement of RNA suitable for bacterial tests and virus tests.

[0002]

2. Description of the Related Art In the field of clinical examination, for example, when it is determined that a bacterial infection is suspected, a sample (specimen) such as blood is obtained from a patient, and the presence of the bacteria is determined by staining and microscopy. After the confirmation, the bacterium in the sample (specimen) is cultured in various media, and the process up to the identification of the bacterium is completed.

[0003] This method can determine even the type of bacteria.
On the other hand, there is a problem that it takes time because culture must be performed.

[0004] Therefore, recently, nucleic acid diagnosis is frequently performed instead of the above-mentioned method. Bacterial nucleic acid diagnosis involves extracting DNA or RNA in a bacterium and detecting the specific nucleotide sequence of the bacterium from the DNA or RNA, thereby identifying the presence or absence of bacterial infection and identifying the bacterial species in a short time. .

[0005] Regarding the nucleic acid diagnosis of bacteria, methods such as dot method, Southern blot method, Northern blot method and the like for fixing nucleic acid to be measured on a filter, in situ methods, and the like.
There are a hybridization method and a liquid layer method. To explain a sandwich assay as an example, first, a nucleic acid fraction is extracted from a bacterium by a conventional method to extract a nucleic acid, the nucleic acid is bound to a nucleic acid probe bound to a solid phase, and then the labeled nucleic acid probe is removed. Contact (solid phase-nucleic acid probe bound to solid phase-nucleic acid to be measured-labeled nucleic acid probe-
B) to form a complex. Next, the labeled nucleic acid probe which is not bound to the nucleic acid to be measured is removed, and finally the label is measured to find out the presence and amount of the nucleic acid to be measured. As the label, for example, an enzyme such as radioisotope, alkaline phosphatase or β-D-galactosidase, a fluorescent substance or a luminescent substance is used.

Further, if at least one of a nucleic acid probe to be bound to a solid phase and a labeled nucleic acid probe is prepared in a manner complementary to a base sequence that the bacteria specifically has, the type of bacteria can be known. It is possible.

[0007]

2. Description of the Related Art In conventional nucleic acid diagnosis, nucleic acids to be measured are genomic DNA, plasmid DNA, messenger RNA, and ribosome RNA.
A is present in only one or a few copies per bacterium, and is unsuitable for highly sensitive nucleic acid diagnosis.

On the other hand, plasmid DNA, messenger RNA, ribosome RNA and the like have a large number of copies per cell and are suitable for highly sensitive measurement. Among them, ribosome RNA has the largest number of copies per cell,
It can realize the most sensitive nucleic acid diagnosis.

For reference, the amounts of nucleic acids in Escherichia coli are listed as follows: genomic DNA 1%, messenger RNA 5%, transfer RNA 15%, ribosomal RNA
79%. Therefore, genomic DNA and ribosomal DNA
For RNA, there is a greater difference in the number of copies of the nucleic acid.

[0010] As described above, in nucleic acid diagnosis, measurement with higher sensitivity can be performed with RNA as compared with DNA. However, RNA
In many cases, the nucleic acid probe itself has a secondary, tertiary or higher order structure, and the nucleic acid probe and the hybridizer
There is a problem that it is difficult to handle. In the hybridization of nucleic acids, the binding of RNA is the strongest.
The binding becomes weaker in the order of NA-RNA binding and DNA binding. Therefore, particularly when DNA is used as a nucleic acid probe, the above-mentioned problem becomes remarkable.

[0011] Among RNAs, transfer RNA and ribosome RNA are known to have particularly complicated three-dimensional structures. For example, ribosome RNA is 5S ribosome RNA (about 120 bases), 16S ribosome RNA.
(About 1500 bases) and 23S ribosomal RNA, and these ribosome RNAs and proteins bind to form a large ribosome.

For this reason, when it is desired to carry out highly sensitive nucleic acid diagnosis, it is preferable to target ribosome RNA having a large number of copies, but it is difficult to hybridize the probe. In the end, the expected measurement sensitivity is not achieved.

[0013]

Means for Solving the Problems The present inventor has focused on the fact that fragmented RNA is unlikely to have a three-dimensional structure, so that the RNA to be measured is alkali-hydrolyzed to have no three-dimensional structure. By doing so, the difficulty of hybridization between RNA and a nucleic acid probe, which was a conventional problem, is solved.
The present invention has been completed.

That is, according to the present invention, the RNA to be measured is fragmented by alkaline hydrolysis, and then the fragmented RNA to be measured and a specific base sequence contained in the RNA to be measured are hybridized with the RNA. Possible DN
It is intended to provide a method for measuring RNA comprising contacting A or RNA probe and measuring the formed hybrid. Hereinafter, the present invention will be described in detail.

In the present invention, the RN directly measured is
A may be prepared, for example, from blood, serum, cells, urine, stool (feces), mucus, sputum and the like obtained from animals and humans according to a usual method. In the present invention, the prepared RNA
Fractionation may be used, and if possible, a ribosomal RNA fraction having a large amount and a large number of copies may be used.
By using the RNA thus purified as a target, the influence of foreign substances can be reduced.

In the present invention, the blood,
A sample containing RNA such as serum or cell lysate may be used directly. When a body fluid or the like is directly used in the present invention, centrifugation or chromatography is performed to obtain R
It is preferable to remove blood cell components, protein components, and the like that may interfere with the hybridization between the NA fragment and the nucleic acid probe.

Examples of the RNA to be measured include RNA encoding a protein specific to bacteria and viruses, and RNA encoding a cancer protein produced by transcription of an oncogene. In other words, the RNA to be measured is RNA that is derived from bacteria, viruses and sometimes oncogenes and is not normally produced, and has a characteristic nucleotide sequence (specification) when compared to normally produced RNA. , Etc.). Therefore, by measuring the presence or absence of such a specific base sequence, it becomes possible to identify not only virus infection, bacterial infection, onset of cancer, etc., but also its type.

On the other hand, even for an RNA which does not encode a specific protein such as rRNA, as described in the Examples below, the measurement of rRNA specific to bacteria such as Escherichia coli will It is possible to detect the presence.

In the present invention, the alkali hydrolysis treatment comprises:
It can be easily carried out by dissolving the prepared RNA fraction in an alkaline solution. PH of alkaline solution is 9-1
What is necessary is just about two. On the other hand, when a bodily fluid or the like is directly used in the present invention, an alkali substance may be directly added thereto.

The degree of fragmentation of the RNA by alkaline hydrolysis depends on the pH, temperature and other contaminants in the solution. As an example, a 40 mM sodium bicarbonate-60 mM sodium carbonate solution (pH 1
0) in a 60 ° C. solution, t = (L0−Lf) / (k ·
L0 · Lf). Here, k = 0.1 / 1 / (k
b · min), L0 = initial length of RNA, Lf = end length of RNA, and t = time in minutes. From this equation, considering, for example, 16S ribosome RNA, when 1500 base (b) of 16S ribosome RNA is added to the above solution, about 570 bases after 30 minutes.
Alkali hydrolysis to about 250 bases after 30 minutes and about 140 bases after 1 hour.

According to the experience of the present inventor, RNA fragmented up to about 250 bases is unlikely to have a three-dimensional structure. Therefore, conditions are determined with reference to the above formula, and further preliminary experiments are performed. What is necessary is just to determine pH, temperature, processing time, etc. at the time of alkali hydrolysis. Even if the fragmentation is performed up to about 250 bases, the possibility that the specific sequence in the RNA to be measured is lost is extremely small. Further, by using about 250 bases, a fragment can be fixed to a filter while preserving a specific base sequence, or, for example, for a sandwich assay, two nucleic acid probes and a hybridizer can be used.
It is also easy to make

The RNA fragmented as described above is subsequently contacted with a nucleic acid probe. The nucleic acid probe may be either DNA or RNA, but considering the strength of hybridization between nucleic acids, RNA
It is preferable that The nucleic acid probe only needs to have a sequence complementary to the specific sequence contained in the RNA to be measured. It is preferable that the specific sequence is a characteristic continuous sequence of about 20 bases. By using a base sequence of this degree or more, the nucleic acid
Can eliminate the risk of non-specific hybridization with the RNA fragment. The specific sequence is not particularly limited, and is not limited as long as it can efficiently measure viral or bacterial infection.

For the specific sequence in the RNA to be measured, for example, the presence or absence of virus infection can be known by selecting a sequence that exists widely in an infectious virus, and a sequence specific to a specific virus can be selected. Then, identification of the infected virus can be achieved simultaneously with the presence or absence of virus infection. Therefore, the specific arrangement may be determined by the practitioner according to the purpose.

As described above, RNA includes messenger RNA, transfer RNA or ribosome RN.
A and other types. Among them, ribosome RNA has the largest amount and the largest copy number. Therefore, in the present invention, it is preferable to use a nucleic acid probe complementary to a specific base sequence present in the ribosome RNA in order to perform highly sensitive measurement.

After the nucleic acid probe is brought into contact with the fragmented RNA to be measured, the formed hybrid is measured by, for example, a technique of high performance liquid chromatography. The fact that it has a large molecular weight as compared with a nucleic acid probe can be used as a clue. However, more simply, a labeling substance such as a radioisotope may be bound to the nucleic acid probe. Above all, substances that can be measured by optical means such as enzymes, fluorescent substances, luminescent substances or absorbing substances (enzymes are produced by adding an enzyme substrate to cause an enzymatic reaction, and optically measuring the enzymatic reaction products generated later. If) is used as a labeling substance, there is no effect on the human body and the measurement itself is simple. The binding between the nucleic acid probe and the label may be performed according to a usual method, and there is no particular limitation.

In the method for measuring RNA of the present invention, the fragmented target RNA may be immobilized on a filter or the like to hybridize the nucleic acid probe, and the measurement may be carried out later. Target RN in a different part
A so-called sandwich assay in which a nucleic acid probe that hybridizes with A is immobilized on an appropriate solid phase and a nucleic acid probe complementary to a specific base sequence is hybridized later may be performed. .

In a sandwich assay, two nucleic acid probes are used. One is a probe for capturing the target RNA, and the other is a probe for measurement bound to a label. Usually, the target probe is captured by immobilizing a nucleic acid probe for capture on a suitable solid phase, and then the probe to be measured is hybridized to measure the RNA to be measured.

However, a nucleic acid probe capable of hybridizing with a specific base sequence in the RNA to be measured is immobilized, and the RNA to be measured is first captured from among the target RNA, and then the other RNA is captured. The RNA is removed and the remaining RNs are detected using a nucleic acid probe conjugated to a label that can subsequently hybridize to a base sequence common to the RNA.
Sometimes all of A is measured.

[0029]

EXAMPLES The present invention will be described below in more detail with reference to Examples, but these Examples do not limit the present invention.

Example 1 (1) Preparation of DNA-immobilized gel 21-mer aminated synthetic DNA, 10 OD (260 n
m indicates that the absorbance is 10. The same applies hereinafter) to D
NA synthesizer (381 manufactured by Applied Biosystems)
After synthesis in A), 2 ml of 1 M phosphate buffer (pH 8.8)
NPR (Toso Co., Ltd.) 100
mg and left for 1 hour with stirring. The base sequence of this DNA is (5 ′)-GCC TTC G
CC ACCGGT ATT CCT- (3 ′), which hybridizes to 16S rRNA of E. coli.
This is a synthetic DNA to be applied.

The mixture was centrifuged to precipitate a gel, and the supernatant was discarded. Then, 3 ml of a 100 mM Tris-HCl buffer (pH 8.0) was added, and the mixture was allowed to stand for 5 minutes with stirring. Unreacted tresyl groups were reacted.

This mixture was further subjected to centrifugation, and the same operation was repeated three times.

The supernatant obtained from the above operation was discarded, and 3 ml of 5 × SSC (3 M NaCl, 0.3 M
Sodium Citrate pH 7.0), 0.5
% Polyvinylpyrrolidone K-30 (manufactured by Nacalai Tesque), 0.5% bovine serum albumin (Chiba Livestock Industry Co., Ltd.)
Res.).

(2) Alkaline phosphatase (AL
P) Preparation of Label-bound DNA 21mer aminated synthetic DNA3OD was synthesized with a DNA synthesizer (381A manufactured by Applied Biosystems), and then 50 µl of 0.1 M sodium hydrogencarbonate, 2 mM
DTA, DMSO (Dimethyl sul
100 μl DSS (Disu) dissolved in
ccinimidyl Subrate, PIER
After the mixture was left at 37 ° C. for 5 minutes, high performance liquid chromatography (HW40, manufactured by Tosoh Corporation) was performed.
F), separated and freeze-dried. The base sequence of this DNA was (5 ′)-ATC TCT ACG CA
TTTC ACC GCT- (3 ′), which is a synthetic DNA that hybridizes to 16S rRNA of E. coli.

50 μl of 3M was added to this DSS-modified synthetic DNA.
Sodium chloride, 0.1 M borate buffer (pH 8.0)
Was added and left at room temperature for 2 hours.

The above solution was subjected to high performance liquid chromatography.
(G3000SWXL, manufactured by Tosoh Corporation)
Synthetic DNA bound to P was obtained.

(3) Measurement of RNA The nucleic acid fraction isolated and dried from Escherichia coli (K12 strain, C600) by the hot phenol method was added with 40 mM sodium hydrogencarbonate and 60 mM sodium carbonate (pH 10.3).
Was added so that the final cell concentration was 10 ⁶ cells / 10 μl, and the mixture was allowed to stand at 60 ° C. for 0, 10, 30 or 60 minutes to carry out an alkaline hydrolysis treatment (0 means that the alkaline hydrolysis treatment was carried out). If not).

The hydrolysis treatment was stopped by adding a neutralizing solution consisting of 1/30 volume of 3M sodium acetate and 1/200 volume of glacial acetic acid.

[0039] 10 µl of the nucleic acid fractionated solution after the completion of the alkaline hydrolysis treatment was mixed with a hybridization solution (5 x S
SC, 0.5% polyvinylpyrrolidone K-30, 0.5
% Bovine Serum Albumin, 1% SDS (Sodium Do
Decyl sulfate (manufactured by Wako Pharmaceutical Co., Ltd.), dextran sulfate (Mw-500000, manufactured by Pharmacia), 1 mM magnesium chloride, 0.1 mM zinc sulfate), and 1 mg of the DNA immobilized gel previously prepared
And left to stand at 50 ° C. for 30 minutes (the stirring operation was performed every 5 minutes).

The mixture was centrifuged, the supernatant was discarded, and the DNA concentration of the hybridization solution was adjusted to 100 ng / DNA.
100 μl of the previously prepared ALP-bound synthetic DNA diluted to a volume of 100 ml was added and left at 50 ° C. for 15 minutes.

Next, the mixture was centrifuged, the supernatant was discarded, and 750 μl of a washing solution (1% Tween 20, 1% dextran sulfate, 0.15 M sodium chloride,
0.015M TEA acetate buffer (pH 8.0), 1m
M magnesium chloride, 0.1 mM zinc sulfate), and the mixture was stirred, centrifuged, and the supernatant was discarded.

After repeating the above washing operation a total of six times,
The gel obtained by discarding the supernatant is mixed with 100 μl of 0.5 M A
MP (aminomethylpropanol) buffer (pH 10.
0) and left at 37 ° C. for 5 minutes.

After adding 100 μl of a 2 mM 4MUP (4 methyl umbelliferone phosphate) solution to the gel-containing solution obtained as described above, the fluorescence intensity was measured using a fluorescence detector. , Its increase rate (ra
te) was calculated.

As a result, when the alkali hydrolysis treatment was not performed, a rate of less than 100 was obtained, whereas when the alkali hydrolysis treatment was performed for 30 minutes or 60 minutes, a rate of 600 or more was obtained. was gotten. The lower limit of detection of Escherichia coli according to this example is about 400 cells, while the lower limit of detection when RNA is not fragmented is 4000 cells, which is 10 times as large. in this way,
A 10-fold increase in sensitivity was achieved by the measurement method of the present invention.

FIG. 1 shows the above results. According to FIG.
It can be seen that the RNA did not have a three-dimensional structure due to the alkali hydrolysis treatment, and it became easy to hybridize with the DNA immobilized on the gel and the DNA bound to ALP.

Further, it is understood that good results can be obtained by subjecting the alkaline hydrolysis treatment to about 30 to 60 minutes for a subject having a small amount of contaminants, such as the nucleic acid fraction targeted in the present example. .

Example 2 Lysis measurement of Escherichia coli 10 μl of a suspension of Escherichia coli at 10 ⁸ cells / ml
Add 1 μl of 6MGdn, 100 mM sodium carbonate buffer (pH 10.0) and add 0, 1, 2, 3,
Leave for 4 or 5 minutes.

To this solution was added 80 μl of a stop solution (37.5).
mM sodium acetate buffer (pH 6.0), 0.19%
(V / v) acetic acid, 5 × SSC) was added to stop the alkaline hydrolysis, and the DNA-immobilized gel prepared in Example 1 was further added, and the mixture was left at 50 ° C. for 30 minutes.

The mixture was centrifuged, the supernatant was discarded, and the DNA concentration of the hybridization solution was adjusted to 100 ng / DNA.
ml of synthetic DN combined with ALP diluted to
A (prepared in Example 1) was added in an amount of 100 μl,
Left for 5 minutes.

Next, the mixture was centrifuged and the supernatant was discarded. 750 μl of the washing solution was added and stirred, and the mixture was further centrifuged and the supernatant was discarded.

After repeating the above washing operation a total of six times,
The gel obtained by discarding the supernatant is mixed with 100 μl of 0.5 M A
MP (aminomethylpropanol) buffer (pH 10.
0) and left at 37 ° C. for 5 minutes.

After 100 μl of a 2 mM 4MUP (4 methyl umbelliferone phosphate) solution was added to the gel-containing solution obtained as described above, the fluorescence intensity was measured using a fluorescence detector. , Its increase rate (ra
te) was calculated.

As a result, when the alkali hydrolysis treatment was not performed, the rate was very small, whereas
Large rate when alkali hydrolysis treatment is performed
was gotten.

FIG. 2 shows the above results. According to FIG.
It can be seen that the RNA did not have a three-dimensional structure due to the alkali hydrolysis treatment, and it became easy to hybridize with the DNA immobilized on the gel and the DNA bound to ALP. As described above, even if the alkali hydrolysis treatment is performed for only a few minutes, the obtained rate greatly changes.

Example 3 Measurement of Lysis of Mycobacterium In the same manner as in Example 1, a 21-mer synthetic DNA was obtained.
Was prepared and immobilized on a gel. This DNA is (5
') -CGG TAT TAG ACC CAGTTT
CCC- (3 '). Further, a 21-mer synthetic DNA was further prepared by the same operation as in Example 1, and AL
Coupled with P. This DNA is (5 ')-AGA C
AT GCA TCC CGT GGT CCT- (3
´). These synthetic DNAs are DNAs that hybridize to Mycobacterium 16S rRNA.

To a cell obtained by centrifuging 1 ml of the culture solution of Mycobacterium bovis BCG, 200 μl of a defatted solution (a mixture of methanol and chloroform) was added.
Treated at 0 ° C. for 2 minutes. Add 300 μl of 100 to this solution
mM sodium carbonate solution (pH 11.0)
Stir and leave at 50 ° C. for 40 minutes.

The solution was neutralized by adding a sodium acetate solution, 75 μl of the supernatant was mixed with 75 μl of the hybridization solution, and the mixture was added to a DNA-immobilized gel and left at 50 ° C. for 30 minutes. did. During this time,
Stirring was performed every 5 minutes.

The above mixture is centrifuged and the supernatant is discarded.
100 ng DNA concentration with hybridization solution
/ D coupled to ALP diluted to / ml
100 μl of NA was added and left at 50 ° C. for 15 minutes.

Next, the mixture was centrifuged, the supernatant was discarded, 750 μl of a washing solution was added, and the mixture was stirred, and the washing operation of centrifuging and discarding the supernatant was repeated a total of 6 times.
The gel obtained by discarding the supernatant is mixed with 100 μl of 0.5 M A
MP (aminomethylpropanol) buffer (pH 10.
0) and left at 37 ° C. for 5 minutes.

After adding 100 μl of a 2 mM 4MUP (4 methyl umbelliferone phosphate) solution to the gel-containing solution obtained as described above, the fluorescence intensity was measured using a fluorescence detector. .

As a result, the lysed RNA (rRNA) of Mycobacterium bovis BCG could be measured.

[0062]

Industrial Applicability According to the present invention, by subjecting RNA to an alkaline hydrolysis treatment to fragment it so as not to have a three-dimensional structure, hybridization between the nucleic acid probe and the RNA to be measured is easily caused. Is what you do.

More specifically, since an RNA fragment of about 250 to 300 bases is unlikely to have a three-dimensional structure, alkali hydrolysis is performed on such a fragment. In particular, in the alkali hydrolysis treatment performed in the present invention, the degree of fragmentation can be arbitrarily determined mainly by adjusting the treatment time, the treatment pH, and the treatment temperature. In addition, it is possible to fragment the target RNA.

The present invention is to measure an RNA to be measured containing a specific base sequence in a target RNA. The specific nucleotide sequence determines the RNA to be measured by other RNs.
A sequence that can be distinguished from A, for example, a sequence that does not exist in human RNA but is found only in bacterial RNA.

The specific base sequence may be destroyed by the alkali hydrolysis treatment. However, the possibility is extremely small if fragmentation is performed to about 250 bases.

RNA has a large copy number as compared with DNA and is contained in a large amount. Therefore, the present invention, which solves the difficulty of hybridization with a nucleic acid probe due to RNA having a three-dimensional structure, is suitable for measurement of bacterial or viral infection in the field of clinical diagnosis and the like. .

[Brief description of the drawings]

FIG. 1 shows the results of Example 1 of the present invention, in which the horizontal axis represents the time during which the alkaline hydrolysis treatment was performed, and the vertical axis represents the rate (the rate of increase in the fluorescence intensity) calculated from the results of the fluorescence measurement. ). When the alkali hydrolysis treatment is 0, that is, when the alkali hydrolysis treatment is not performed, only a small rate of less than 100 can be obtained, but when the hydrolysis treatment is performed, a larger rate can be obtained. The fact that the rate is large means that A
This indicates that LP is contained in a large amount. ALP is bound to the synthetic DNA, and is bound to the synthetic DNA immobilized on the gel via the RNA to be measured.
Therefore, the fact that the amount of ALP was increased by the alkaline hydrolysis treatment means that the RNA to be measured and the synthetic DNA immobilized on the gel or the synthetic DNA bound to ALP were used.
Indicates that both hybridizations became easier to occur.

FIG. 2 shows the results of Example 2 of the present invention. The horizontal axis represents the time during which the alkaline hydrolysis treatment was performed, and the vertical axis represents the rate calculated from the results of the fluorescence measurement.
This figure also shows that the measured rate was higher when the alkali hydrolysis treatment was performed than when the alkali hydrolysis treatment was not performed.
It can be seen that the hybridization between the RNA to be measured by alkaline hydrolysis and the synthetic DNA was liable to occur.

Claims (3)

(57) [Claims] 1. An RNA having a three-dimensional structure to be measured is subjected to alkaline hydrolysis treatment to obtain an R of about 250 to 300 bases.
Fragmented into NA fragments, and then the fragmented R
DNA or RNA capable of hybridizing with NA and a specific base sequence contained in the RNA to be measured
A method for measuring RNA, comprising contacting a probe and measuring a formed hybrid. 2. The method according to claim 1, wherein the RNA is fragmented by alkaline hydrolysis in a solution having a pH of 9 to 12. 3. The RNA to be measured is a ribosomal RNA.
The method according to claim 1 or 2, wherein