JPH10229899A - Primer for detecting bcr/abl type chimera messenger rna, and detection of bcr/abl type chimera messenger rna and using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new primer useful for diagnosing leukemia, suitable for detecting a BCR/ABL type chimera mRNA having a restriction site on Major-bcr by NASBA method, comprising a nucleic acid having a specific base sequence and a prescribed number of bases. SOLUTION: This primer comprises a nucleic acid having a base sequence composed of continuous 15-40 bases among base sequences of formula I and formula II, is a new primer at a forward side having a restriction site at Major- bcr of formula 1 and at a reverse side at which an RNA polymerase promoter is bonded to a partial sequence of formula II, is useful for detecting a BCR/ABL type chimera mRNA resulting from the mutual translocation of 9 chromosome and 22 chromosome, is detected in a case of a disease of >=90% of chronic myelogenic leukemia(CML) and about 20% of acute lymphocytic leukemia(ALL) and is useful for diagnosing CML and ALL. The primer is obtained by selecting a sequence capable of detecting the chimera mRNA from a BCR gene of 22 chpomosome and a c-abl gene of 9 chromosome in high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a BCR / ABL chimera m
The present invention relates to an RNA detection primer and a method for detecting a BCR / ABL chimeric mRNA using the same. The present invention relates to chronic myeloid leukemia (hereinafter sometimes referred to as "CML") and acute lymphocytic leukemia (hereinafter sometimes referred to as "ALL").
Useful for diagnosis of

[0002]

2. Description of the Related Art The Philadelphia (Ph) chromosome contains 9 chromosomes.
A chromosome resulting from the translocation t (9; 22) (q34: q11) between chromosome 22 and chromosome 22, which is detected in more than 90% of CML cases and about 20% of ALL cases. . During this translocation, the c-abl gene on chromosome 9 is cleaved upstream, and fused with the BCR gene on chromosome 22.
A BCR / ABL-type chimeric gene is formed (Himaru Hisamaru, Experimental Medicine Vol. 9 No. 10 (extra) 1991 pp. 137-142).

There are roughly two types of breakpoints at which the translocation of the Ph chromosome occurs.
r ") and minor-bcr (hereinafter" m-bc
r "). BCR / ABL-type chimeric genes having a breakpoint in M-bcr are detected in 90% or more of CML. ALL with Ph translocation is about 20%
Approximately half detect a BCR / ABL-type chimeric gene having a breakpoint in M-bcr, and the other half detect a BCR / ABL-type chimeric gene having a breakpoint in m-bcr.

[0004] The BCR / ABL type chimeric gene is transcribed into BCR / ABL.
This produces an ABL-type chimeric mRNA. Therefore, it is useful for the diagnosis of CML and ALL to detect a BCR / ABL-type chimeric mRNA and to determine whether it has a breakpoint in M-bcr or whether it has a breakpoint in m-bcr.

[0005] Conventionally, BCR / ABL type chimeric mRNA has been
It is amplified and detected by the CR method (Japanese Unexamined Patent Publication No.
No. 96; Proc. Natl. Acad. Sci. USA Vol. 85, pp.
569804702, August 1988 Medical Sciences). The conventional method using the RT-PCR method requires two to three days for measurement, and the operation is complicated. Although the sensitivity is good, it goes without saying that a more sensitive measuring method is more preferable.

[0006]

Accordingly, an object of the present invention is to provide a means for detecting a BCR / ABL type chimeric mRNA with a shorter time, a simpler operation, and a higher sensitivity than the conventional method. To provide.

[0007]

Means for Solving the Problems The present inventors have proposed a BCR / AB
L-type chimeric mRNA was analyzed using NASBA (nuclei
c acid sequence-based amplification) method (aka “3S
R ”(Self-sustained sequence replication) method (E. Fah
y et al., PCR Methods and Applications, pp. 25-32,
1991, Cold Spring Harbor Laboratory Press ISSN 105
An attempt was made to achieve the above object by amplifying according to 4-9803), but the sensitivity was insufficient. However, as a result of diligent research, it was found that BCR / ABL-type chimeric mRN
A primer for NASBA capable of detecting A was found, and the present invention was completed.

[0008] That is, the present invention relates to SEQ ID NO: 1
15 to 40 consecutive nucleotides in the base sequence represented by
Major-bc consisting of a nucleic acid having a base sequence consisting of bases
NASBA of BCR / ABL type chimeric mRNA having a cleavage point at r
Provide a forward primer for detection by the method. The present invention also has a breakpoint in minor-bcr consisting of a nucleic acid having a continuous base sequence of 20 to 24 bases in the base sequence represented by SEQ ID NO: 3 in the sequence listing.
Provided is a forward primer for detecting BCR / ABL type chimeric mRNA by NASBA method. Furthermore, the present invention relates to a nucleotide sequence represented by SEQ ID NO: 5 in the sequence listing.
Provided is a reverse primer for detecting a BCR / ABL-type chimeric mRNA by the NASBA method, comprising a nucleic acid having a nucleic acid fragment having a base sequence of 40 to 40 bases and an RNA polymerase promoter sequence linked thereto. Further, the present invention provides a method using the forward primer of the present invention and the reverse primer of the present invention to obtain a Ma
amplifying the antisense RNA of the BCR / ABL type chimeric mRNA having a breakpoint at jor-bcr or minor-bcr, and detecting the amplified RNA;
Provided is a method for detecting a BCR / ABL chimeric mRNA having a breakpoint in r-bcr.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION BCR / ABL Having a Break Point at M-bcr
A forward-side primer (sense primer) for detection of a type-type chimeric mRNA (hereinafter sometimes referred to as “M-bcr chimeric mRNA”) by the NASBA method comprises 15 consecutive nucleotides represented by SEQ ID NO: 1 in the sequence listing. Base or 4
It consists of a nucleic acid having a base sequence consisting of 0 bases. Among them, a DNA primer having a sequence consisting of 20 bases represented by SEQ ID NO: 2 in the sequence listing is particularly preferred. In addition, this DNA primer hybridizes, M-bc
r The position in the chimeric mRNA is shown in FIG. 1 (indicated by “2F2”).

A forward primer (sense primer) for detecting a BCR / ABL-type chimeric mRNA having a cleavage point at m-bcr (hereinafter sometimes referred to as “m-bcr chimeric mRNA”) by the NASBA method is shown in the sequence listing. It consists of a nucleic acid having a base sequence consisting of consecutive 20 to 24 bases of the base sequence represented by SEQ ID NO: 3. Of these,
A DNA primer having a sequence consisting of 20 bases represented by SEQ ID NO: 4 in the sequence listing is particularly preferred. Note that this D
M-bcr chimera m to which NA primer hybridizes
The position in the RNA is shown in FIG. 2 (indicated by 1F12).

M-bcr chimeric mRNA and m-bcr chimera m
The reverse primer (antisense primer) for RNA measurement (common to both) is a nucleic acid fragment having a continuous base sequence of 15 to 40 bases among the base sequence represented by SEQ ID NO: 5 in the sequence listing, Consists of a nucleic acid linked to an RNA polymerase promoter sequence. RN
It is particularly preferable that the base sequence other than the A polymerase promoter sequence has a sequence consisting of 20 bases represented by SEQ ID NO: 6 in the sequence listing. The RNA polymerase promoter sequence is not particularly limited, and may be a double-stranded DNA
Any template may be used as long as it is a promoter sequence capable of binding mRNA polymerase capable of transcribing mRNA using as template. Preferred examples include the promoter sequence of T7 phage represented by SEQ ID NO: 7. Therefore, as the reverse-side primer, 47 shown in SEQ ID NO: 8 in the sequence listing was used.
DNA primers having a sequence consisting of bases are particularly preferred. The positions in the M-bcr chimeric mRNA and m-bcr chimeric RNA where the DNA primer hybridizes are shown in FIG. 1 (indicated by “2R2”) and FIG.
R2 ").

M-bcr chimeric mRNA or m-bcr using the forward primer and the reverse primer of the present invention.
The bcr chimeric mRNA can be detected by amplifying the antisense RNA by the NASBA method using these primers and detecting the amplified antisense RNA. Specifically, it can be performed as follows.

First, RNA in a specimen cell is extracted. The sample cells may be any cells expressing the BCR / ABL type chimeric gene. Examples of preferable sample cells include leukocytes (whole blood can be used as a sample),
Bone marrow cells (bone marrow fluid can be used as a specimen) and the like can be mentioned, but are not limited thereto. MRNA in cells can be extracted by a well-known method using an oligo dT column. Alternatively, all nucleic acids may be extracted by a conventional phenol method. After lysing the cells with a lysis buffer and binding the eluted nucleic acids to the silica particles, washing is repeated to repeat the nucleic acids (RNA and DN).
A) Boom extraction method to separate both (R. Boom et al., JOURN
AL OF CLINICAL MICROBIOLOGY, Vol. 28, No. 3, Mar.
1990, p. 495-503), RNA and DNA may be extracted.

Next, using the extracted nucleic acid as a template,
Perform the NASBA method. In the NASBA method, in addition to the above-mentioned forward primer and reverse primer, reverse transcriptase, RNase, RNA polymerase, DNA polymerase, and four types of deoxyribonucleotides and four types of ribonucleotides used as raw materials for nucleic acids are used. In addition, it is preferable to use a reverse transcriptase having a DNA-dependent DNA polymerase activity as in the following examples, because one enzyme can be used as the reverse transcriptase and the DNA polymerase. The preferred concentration range of each component in the reaction solution is 0.01-100 U / μl for reverse transcriptase and 0.0001-0.1 U / μ for RNaseH.
1, RNA polymerase is 0.1 to 100 U / μl, and each nucleotide is about 0.1 to 100 mM. The NASBA method basically involves only incubating a reaction solution containing these components and a test nucleic acid at around 41 ° C., which is the optimal temperature for the reaction. The incubation time is not particularly limited, but is usually about 60 to 120 minutes, preferably 8 to 120 minutes.
It is about 0 to 100 minutes. In addition, M-bcr chimeric mRNA
Is detected, use M-
Using primers for bcr chimeric mRNA detection, m-bcr
When detecting chimeric mRNA, a primer for detecting m-bcr chimeric mRNA is used as a forward primer. The reverse primer is common.

If the sample nucleic acid contains a BCR / ABL type chimeric RNA, the reverse primer first hybridizes to the BCR / ABL type chimeric RNA. The reverse primer has an RNA polymerase promoter sequence as described above. However, since there is no complementary portion in the template RNA as well as this promoter sequence portion, hybridization does not occur, and hybridization occurs only in portions other than the promoter sequence. After the reverse primer has hybridized, cDNA (antisense) complementary to the template RNA is synthesized by the action of reverse transcriptase, while the template RNA that has formed a double strand with the DNA is degraded by the action of RNase. , The cDNA becomes single-stranded. This cDNA has an RNA polymerase promoter sequence at the end because the reverse primer has an RNA polymerase promoter sequence. Next, the forward primer is cD
It hybridizes with NA and forms double-stranded DNA by the action of DNA polymerase. This double-stranded DNA is
Since it has an RNA polymerase promoter sequence at its end, RNA polymerase binds to this portion to cause transcription, and antisense RNA is produced one after another. Then
The forward primer hybridizes to the produced antisense RNA, and the RNA-D
An NA double strand is formed, and the RNA strand of the double strand is degraded by the action of RNase to become a single-stranded cDNA (sense). The reverse primer hybridizes to this single-stranded DNA, and the double-stranded DN
A is synthesized. As described above, this double-stranded DNA
The NA polymerase binds to produce antisense RNA one after another. By repeating this cycle, an antisense RNA complementary to the template RNA is amplified.

The antisense R thus amplified
By detecting NA, it is possible to know whether or not the test nucleic acid contains the M-bcr chimeric mRNA or the m-bcr chimeric mRNA. Amplified antisense RNA
Can be detected by a conventional method such as electrophoresis or sandwich hybridization. In the sandwich hybridization method, a capture DNA probe that hybridizes with a region in the antisense RNA to be detected is bound to a solid phase such as a well of a plate, and this is reacted with a sample to capture the antisense RNA on the solid phase. A method of detecting a target antisense RNA by washing and reacting a labeled probe that hybridizes with another region of the antisense RNA, and measuring the label of the labeled probe captured on the solid phase after washing. It is. The label is not particularly limited, and an enzyme label, a fluorescent label, a radiolabel, and the like can be used. It should be noted that an apparatus for performing the sandwich hybridization method is known (Japanese Journal of Clinical Laboratory Automation Vol. 20, No. 5, 728-731, 199).
5), it is possible to carry out using such a known device.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

Example 1 Detection of M-bcr Chimeric mRNA RN in K-562 (commercially available from Dainippon Pharmaceutical Co., Ltd.), a cell derived from a human chronic myeloid leukemia patient containing M-bcr chimeric mRNA
A, and extract the RNA in pure water (which may be a commonly used TE buffer) at 100 ng / μl,
0 ng, 1 ng, 100 pg, 10 pg, 1 pg or 1
First, add 5 μl of a solution containing
Mix with 10 μl of SBA composition solution (including primers, each ribonucleotide, each deoxyribonucleotide, etc.)
65 ° C for RNA denaturation and primer annealing
For 5 minutes and then at 41 ° C. for 5 minutes. Next, the enzyme mixture (reverse transcriptase (AMV-
RT), T7 RNA polymerase, RNaseH) (5 μl), gently mixed and centrifuged, and incubated at 41 ° C. for 90 minutes. The forward primer used was a DNA having the base sequence of SEQ ID NO: 2, and the reverse primer was a DNA having the sequence of SEQ ID NO: 8. Each primer was chemically synthesized using a commercially available synthesizer. The final concentration of each reagent is as follows.

Tris (pH 8.5) 40 mM MgCl ₂ 12 mM KCl 70 mM DTT 5 mM dNTPs (each) 1 mM NTPs (each) 2 mM RNaseH 0.1 units / tube T7 RNA polymerase 40.0 units / tube AMV-RT 8.0 units / tube

The obtained reaction solution was diluted 10-fold, and the amplified antisense RNA was detected using a known sandwich hybridization device (DNA automatic detection device “Mark I” manufactured by Toyobo Co., Ltd.). As a capture probe, a DNA probe having the base sequence of SEQ ID NO: 9 in the sequence listing was used, and as a labeling probe, a DNA probe having the base sequence of SEQ ID NO: 10 in the sequence listing was used. The label of the labeled probe was alkaline phosphatase, and the detection of the label was performed by reacting a 1,2-dioxetane compound as a luminescent substrate and detecting the generated luminescence. The region where the capture probe and the labeled probe hybridize is shown in FIG.
ure probe (plate) "and" Detection probe ").

On the other hand, for comparison, the already established R
Detection was performed using the same specimen by the T-PCR method.
The results are shown in Table 1 below.

[0022]

Table 1 shows that the detection sensitivity of the method of the present invention is about 10 times higher than that of the conventional RT-PCR.

Comparative Example 1 D hybridizing with the region indicated by 2F1 in FIG.
NA was used as a forward primer, and 2 in FIG.
The M-bcr-type chimeric mRNA was detected in the same manner as in Example 1 using DNA in which the T7 RNA polymerase promoter sequence represented by SEQ ID NO: 7 was bound to the DNA hybridizing with the region represented by R1 as the reverse primer. . As a result, the detection sensitivity was about 1/100 of that in Example 1.

Example 2 Nucleic acid was extracted by a Boom method from bone marrow fluid or peripheral blood of 60 patients with chronic myeloid leukemia and 40 healthy persons, and the measurement was carried out in the same manner as in Example 1. When the cutoff value was 5,000 cps, 55 cases were positive and 45 cases were negative.

Example 3 A cell derived from a human acute lymphoblastic leukemia patient producing m-bcr type chimeric mRNA was used as a material, a nucleic acid having the nucleotide sequence of SEQ ID NO: 4 was used as a forward primer, and a reverse primer was used. The same operation as in Example 1 was performed using the same primer as in Example 1. Note that the same capture probe and label probe as in Example 1 were used. The region where the capture and labeled probes hybridize is shown in FIG. Table 2 shows the results.

[0027]

Comparative Example 2 1F1, 1F2, 1F3, 1F3S, 1F4, 1F1, 1F2, 1F3, 1F4
1F4S, 1F6, 1F7, 1F8, 1F9, 1F10, 1F11 and 2R1, and 2R
Various combinations of 3, namely 1F1-2R3, 1F2-2R3, 1F
3-2R3, 1F3S-2R3, 1F4-2R3, 1F4S-2R3, 1F6-2R3, 1F7-2
R3, 1F8-2R3, 1F9-2R3, 1F10-2R3, 1F11-2R3, 1F12-2R
The same operation as in Example 3 was performed using 3, 1F4-2R1, 1F6-2R1, 1F10-2R1, and 1F11-2R1. As a result, each of the combinations has a detection sensitivity of 1/10 to 1 / compared to the case of Example 3.
It was around 1000.

[0029]

According to the present invention, the BCR / ABL type chimeric mR
The effect is obtained that the NA can be detected with higher sensitivity than the conventional method and with a simple operation in a short time.

[0030]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 40 Sequence type: nucleic acid sequence CGTGTGTGAA ACTCCAGACT GTCCACAGCA TTCCGCTGAC 40

SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid sequence ACTCCAGACT GTCCACAGCA 20

SEQ ID NO: 3 Sequence length: 24 Sequence type: nucleic acid sequence CCGGGCAGAT CTGGCCCAAC GATG 24

SEQ ID NO: 4 Sequence length: 20 Sequence type: nucleic acid sequence GGGCAGATCT GGCCCAACGA 20

SEQ ID NO: 5 Sequence length: 40 Sequence type: nucleic acid sequence AATACGTTAG AGTGTTATCT CCACTGGCCA CAAAATCATA 40

SEQ ID NO: 6 Sequence length: 20 Sequence type: nucleic acid sequence AGTGTTATCT CCACTGGCCA 20

SEQ ID NO: 7 Sequence length: 27 Sequence type: nucleic acid sequence AATTCTAATA CGACTCACTA TAGGGAG 27

SEQ ID NO: 8 Sequence length: 47 Sequence type: nucleic acid sequence AATTCTAATA CGACTCACTA TAGGGAGAGT GTTATCTCCA CTGGCCA 47

SEQ ID NO: 9 Sequence length: 20 Sequence type: nucleic acid sequence TGAAGCCGCT CGTTGGAACT 20

SEQ ID NO: 10 Sequence length: 24 Sequence type: nucleic acid sequence TAGCATCTGA CTTTGAGCCT CAGG 24

[Brief description of the drawings]

FIG. 1 is a diagram showing a base sequence near a Major-bcr cleavage point of a BCR / ABL-type chimeric gene and regions to which various primers and probes hybridize.

FIG. 2 is a view showing a base sequence near a minor-bcr cleavage point of a BCR / ABL type chimeric gene and a region where various primers and probes hybridize.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Masaya Segawa 2-1-1 Katata, Otsu-shi, Shiga Prefecture Inside Toyobo Co., Ltd. (72) Inventor Hiroshi Takarada 2-1-1 Katata, Otsu-shi, Shiga Prefecture Toyobo Co., Ltd.

Claims (11)

[Claims] 1. A major-bcr having a breakpoint consisting of a nucleic acid having a continuous base sequence of 15 to 40 bases in the base sequence represented by SEQ ID NO: 1 in the sequence listing.
Forward primer for detection of BCR / ABL type chimeric mRNA by NASBA method. 2. The primer according to claim 1, which has a base sequence represented by SEQ ID NO: 2 in the sequence listing. 3. A minor-bcr consisting of a nucleic acid having a continuous base sequence of 20 to 24 bases out of the base sequence represented by SEQ ID NO: 3 in the sequence listing, having a breakpoint at minor-bcr.
Forward primer for detection of BCR / ABL type chimeric mRNA by NASBA method. 4. The primer according to claim 3, which has a base sequence represented by SEQ ID NO: 4 in the sequence listing. 5. A BCR / ABL comprising a nucleic acid having an RNA polymerase promoter sequence linked to a nucleic acid fragment having a continuous nucleotide sequence consisting of 15 to 40 nucleotides in the nucleotide sequence represented by SEQ ID NO: 5 in the sequence listing. Type chimeric mR
Reverse side primer for detection of NA by NASBA method. 6. The primer according to claim 5, wherein the base sequence other than the RNA polymerase promoter sequence is represented by SEQ ID NO: 6 in the sequence listing. 7. The primer according to claim 5, wherein the RNA polymerase promoter sequence is a T7 phage RNA polymerase promoter sequence. 8. The primer according to claim 7, wherein the RNA polymerase promoter sequence has a base sequence represented by SEQ ID NO: 7 in the sequence listing. 9. The primer according to claim 8, which has a base sequence represented by SEQ ID NO: 8 in the sequence listing. 10. A major-bc method using the forward primer according to claim 1 or 2 and the reverse primer according to any one of claims 5 to 9 by the NASBA method.
A method for detecting a BCR / ABL chimeric mRNA having a breakpoint at Major-bcr, comprising amplifying an antisense RNA of a BCR / ABL-type chimeric mRNA having a breakpoint at r and detecting the amplified RNA. 11. A minor-bc by the NASBA method using the forward primer according to claim 3 or 4, and the reverse primer according to any one of claims 5 to 9.
A method for detecting a BCR / ABL-type chimeric mRNA having a breakpoint at minor-bcr, comprising amplifying an antisense RNA of a BCR / ABL-type chimeric mRNA having a breakpoint at r and detecting the amplified RNA.