JPH04267895A - Simple method for detecting dna product amplified by polymerase chain reaction(pcr) - Google Patents

Abstract

PURPOSE:To enable high-sensitivity detection of a DNA product for the clinical examination capable of extremely easily treating a number of specimens in a short time by amplifying a DNA product by polymerase chain reaction(PCR) and reacting the amplified product with a fluorescent substance having affinity to DNA. CONSTITUTION:The objective DNA to be detected (e.g. human hepatitis C virus DNA) is amplified by a polymerase chain reaction(PCR) comprising the repeated DNA synthesis reaction with a DNA polymerase using two kinds of primers holding the DNA region therebetween. The amplified objective DNA product is added and reacted to a solution produced by dissolving a fluorescent substance having affinity to DNA (e.g. mithramycin) in 300mM aqueous solution of magnesium chloride. The intensity of the fluorescence is measured at an excitation wavelength of 440nm and a detection wavelength of 540nm to easily detect the objective DNA product in high sensitivity.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to polymerase chain reaction (
This invention relates to a simple method for detecting DNA products amplified by PCR.

0002

[Prior Art] Polymerase chain reaction (PCR) uses two types of primers that sandwich a DNA region of interest.
This is a method in which the desired DNA fragment is amplified one million times or more by repeating the DNA synthesis reaction using NA polymerase. Therefore, since the PCR method is in principle a highly sensitive detection method that can amplify even one copy of DNA, it is expected to be applied to clinical diagnostics such as virus detection, cancer diagnosis, and genetic disease diagnosis.

However, detection of the PCR reaction product generally requires agarose or polyacrylamide electrophoresis followed by ethidium bromide staining, as well as observation and photography of the reaction product by UV irradiation. This method is difficult to operate, requires a limited number of samples to be processed, and is time-consuming, making it difficult to use in general hospitals and clinical laboratories. Therefore, although the diagnostic method using PCR method is highly useful, the current situation is that the method for detecting the reaction product is an obstacle and is not widely used.

0004

[Problems to be Solved by the Invention and Objects of the Invention] According to conventional techniques, a simple detection method for PCR reaction products that can process multiple samples at once has not yet been established.

[0005] The present invention has been made in view of these difficulties in the prior art, and the main purpose of the present invention is to easily and quickly detect a large number of samples in the detection of DNA products propagated by the PCR method. The purpose is to provide a method that can be processed all at once.

[0006]

[Means and effects for solving the problems and achieving the objects] As a result of extensive research into methods for detecting PCR reaction products, the present invention has revealed that if a fluorescent substance that has an affinity for DNA is used,
We have discovered that the reaction product can be easily detected by combining a PCR reaction product with a fluorescent substance and measuring the resulting fluorescence, thereby solving the problems of the prior art and achieving the above objectives. Ta. In the detection method according to the present invention, the DNA of the PCR reaction product is DNA or RNA derived from organisms, viruses, plasmids, and genetic recombinants.
can be used as a template, and antibiotics, nuclear staining dyes, anticancer drugs, etc. can be used as fluorescent substances that have affinity for DNA. These fluorescent substances include:
For example, mithramycin, acridine orange, ethidium bromide, 4',6'-diamidino-2-phenylindole, quinacrine mustard, etc. can be used. In particular, mithramycin and ethidium bromide are effective for fluorescent substances that have an affinity for DNA.

[0007] The present invention will be explained in more detail below with reference to Examples. In addition, although the present invention example discloses a method using mithramycin and ethidium bromide,
If it is a fluorescent substance that has an affinity for DNA, P
CR products can be detected, and the present invention is not limited to the examples.

[0008]

[Example] (1) Example of detection of PCR product using mithramycin PCR using human hepatitis C virus (HCV) PCR
This will be explained based on an example of product detection. Using two sets of HCV-specific primers, PCR was performed twice consecutively using serum from a hepatitis C patient and a normal person. The PC obtained at this time
The results of confirming the R product by polyacrylamide gel electrophoresis are shown in FIG. As shown in FIG. 1, a band was confirmed at the predicted 180 base pair position in the serum of a hepatitis C patient. On the other hand, no band was detected in serum from normal subjects. Using these two specimens, the following reaction with a fluorescent substance was performed.

[0009] To 20 μl of the above PCR reaction solution, add 3 mL of distilled water.
After adding 10 μl of 200 μg/ml mithramycin dissolved in 70 μl and 300 mM magnesium chloride solution, the fluorescence intensity was measured at an excitation wavelength of 440 nm and a detection wavelength of 540 nm. The results are shown in Table 1.

Table 1 PCR with mithramycin
Detection of reaction products

Fluorescence intensity


HCV patient serum (PCR positive)
1.5±0.2
Normal human serum (PCR negative)
0.4±0.1
Blank (primer only)
0.4±0.1 Here, PCR positivity is about 3 to 4 times the fluorescent intensity of negative,
From this, if the blank fluorescence intensity is set as a cutoff value, it can be determined that anything exhibiting a fluorescence intensity higher than this value contains a PCR product.

(2) Example of Detection of PCR Products Using Ethidium Bromide Measurements were carried out using the two PCR reaction positive and negative samples shown in FIG. After adding 370 μl of distilled water and 10 μl of an ethidium bromide solution dissolved in distilled water at a concentration of 20 μl/ml to 20 μl of the above PCR reaction solution, the fluorescence intensity was measured at an excitation wavelength of 260 nm and a detection wavelength of 450 nm. The results are shown in Table 2.

Table 2 P by ethidium bromide
Detection of CR products

Fluorescence intensity


HCV patient serum (PCR positive) 12.1±1.3
Normal human serum (PCR negative)
5.7±0.8
Blank (primer only)
5.4±0.5 Here, PCR positivity is approximately twice the fluorescence intensity of negative PCR, so if the blank fluorescence intensity is set as the cutoff value, anything showing a higher fluorescence intensity will be detected. It can be determined that it contains a PCR product.

[0013]

According to the PCR product detection method of the present invention, conventional operations such as electrophoresis, ethidium bromide staining, confirmation of reaction products by UV irradiation, and photography are not required at all. Therefore, when detecting PCR reaction products, the sample is mixed with a fluorescent substance that has an affinity for DNA.
Since the measurement is completed simply by measuring the fluorescence specifically produced by the DNA product, the operation is simplified and the measurement time can be significantly shortened. Furthermore, the method of the present invention makes it possible to process a large amount of specimen at once, and because it is simple, automatic or semi-automated detection is possible.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing polyacrylamide gel electrophoresis of a PCR product of human hepatitis C virus used in the present invention.

Claims (3)

[Claims] 1. A simple method for detecting DNA, which comprises reacting a product amplified by polymerase chain reaction (PCR) with a fluorescent substance that has an affinity for DNA. Claim 2: Claim (1) characterized in that the product (DNA) amplified by polymerase chain reaction (PCR) uses DNA or RNA derived from an organism, virus, plasmid, or genetic recombinant as a template. ) DN
A simple detection method for A. 3. The simple method for detecting DNA according to claim 1 or 2, wherein the fluorescent substance showing affinity for DNA is an antibiotic, a dye for nuclear staining, or an anticancer drug.