JP2015023822A - Viral test method, viral test device, and well plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a viral test method, etc. capable of measuring even a low-concentration virus precisely, and further having excellent repeatability.SOLUTION: When a given number of times of PCR reactions are completed, a given wavelength light from a light source 15 is irradiated. A well 5 which is irradiated with the light source is divided into the well in which a fluorescence is generated and the well in which a fluorescence is not generated. A fluorescent emission well 6a is a well in which a nucleic acid is present when dispensing a nucleic acid extract into the well 5. When one nucleic acid is present in the well 5, the nucleic acid is increased by PCR reaction. Accordingly, this reaction progress amplifies fluorescence. An analyzing part counts a number of the wells of the fluorescent emission well 6a with the virus of a measuring object or a fluorescence nonluminescent well 6b, and calculates the number of wells in which initially the nucleic acid was present.

Description

  The present invention relates to a virus inspection method and a virus inspection apparatus capable of accurately quantifying a viral gene such as hepatitis virus, and a well plate used therefor.

  The PCR (Polymerase Chain Reaction) method is known as a method for amplifying a target DNA region from a very small amount of DNA in a short time. The principle uses two known sequences on double-stranded DNA to design two primer DNAs that face each other in the synthesis direction. When the DNA in the region sandwiched between these primers is synthesized, it is amplified as a template in the next cycle. By repeating such a reaction, the DNA strand can be amplified geometrically.

  By using such a PCR method, virus diagnosis in serum can be performed. Here, a virus is composed of a protein called capsid and a nucleic acid, usually DNA or RNA, and cannot be propagated alone without cytoplasm. For example, in a test for HBV (hepatitis B virus), a certain level of how much HBV is present in serum can be measured. In order to measure the concentration of how much HBV is present in the serum, a fluorescent primer is attached to the HBV viral DNA, and the DNA is amplified by the PCR method to measure the increase or decrease in fluorescence intensity. The method is used. RNA in HCV virus can also be measured by the same method.

  In this method, first, virus is lysed and extracted from serum, and HVB DNA is extracted therefrom. Next, specific DNA in HBV is amplified. For this purpose, primers suitable for HBV-DNA are designed to amplify HBV-DNA. In particular, when the concentration of HBV-DNA is low, it cannot be measured unless it is increased. At this time, specific fluorescence is imparted to the primer, and the fluorescence intensity increases or attenuates in proportion to the increase in HBV-DNA, whereby the concentration of HBV-DNA can be estimated. The increase in HBV-DNA depends on the PCR method, but theoretically doubles at one time. When an appropriate primer is designed for HBV-DNA, fluorescence is amplified or attenuated when PCR is performed about 40 times, so that the fluorescence intensity is such that HBV-DNA can be detected.

  FIG. 10 is a conceptual diagram showing the relationship between the PCR cycle by the conventional PCR method and the fluorescence intensity. For example, when a fluorescent increase indicator that amplifies fluorescence according to the PCR cycle is used, the fluorescence intensity is amplified as the PCR cycle increases as shown in FIG.

  Here, L to P in FIG. 10 are due to a difference in the copy number of the nucleic acid of the virus in the sample, and the copy number of the nucleic acid in the virus in the sample is L> N> O> P. Here, the copy number represents the number of nucleic acids such as DNA and RNA. In the present application, in some descriptions, there is a portion described as the virus copy number for the sake of simplicity, but this is synonymous with the virus nucleic acid copy number. As shown in the figure, the tendency of the fluorescence increase by the PCR cycle varies depending on the number of virus copies originally present per ml. For example, when the copy number is relatively large, the amplification of the fluorescence is confirmed even with a few PCR cycles as in the case of the L line. For example, after 40 cycles, the fluorescence is amplified to some extent. On the other hand, when the copy number is small, the amplification of fluorescence is not confirmed unless a certain number of PCR cycles elapse as in the case of P-line.

  In this way, it is considered that the DNA in the specimen is also amplified at the same rate as the standard solution by measuring the fluorescence intensity after 40 cycles, for example, using a standard solution with a clear copy number in advance. Therefore, based on the result of the standard solution, the number of virus copies in the original specimen can be estimated.

  Thus, as a method for predicting the copy number from the relationship between the number of PCR cycles and the fluorescence intensity, there is a method in which a nucleic acid probe is hybridized to a target nucleic acid and the amount of change in emission of the fluorescent dye before and after hybridization is measured. Yes (Patent Document 1).

JP 2002-191372 A

  However, the conventional method is not sufficient in measurement accuracy particularly at a low copy number due to problems such as variation in measurement of fluorescence intensity. For example, in an apparatus that is currently in practical use, the copy number measurement lower limit is about 125 copies / ml, and those lower than this are difficult to detect and quantify. However, for example, in order to detect hepatitis patients early and start early treatment, a method for accurately detecting the nucleic acid concentration in a diluted virus is required.

  Further, when the relationship between the PCR cycle and the fluorescence intensity is obtained as shown in FIG. 10, the reproducibility of the fluorescence amplification with respect to the PCR cycle is not necessarily good. For this reason, for example, even if the number of copies is 1000 copies / ml and 10,000 copies / ml, it is possible to accurately distinguish between 1000 copies / ml and 1500 copies / ml. It was difficult.

  The present invention has been made in view of such problems, and it is possible to accurately measure a nucleic acid extract containing a nucleic acid extracted by processing a low-concentration virus, and has excellent reproducibility. The purpose is to provide inspection methods.

  In order to achieve the above-mentioned object, the first invention is a virus inspection method, using a well plate having a plurality of wells whose inner surfaces are hydrophilic, filling the well with a nucleic acid extract, and sealing the well In this state, the PCR reaction is performed a predetermined number of times, the number of the wells in which the fluorescence is generated or the number of the wells in which the fluorescence has disappeared is counted, and the number of virus copies per predetermined amount of the sample liquid is obtained from the total volume of the wells A virus inspection method characterized by:

  In this way, the number of wells in which fluorescence has been generated or disappeared is counted by fluorescence amplification or decay by PCR method so that the nucleic acid of the nucleic acid extract extracted from the virus enters one well. Thus, the copy number of the nucleic acid in the virus can be quantified with high accuracy. That is, unlike conventional methods, the quantification is not based on the fluorescence intensity in one well, and therefore, it is not affected by variations in fluorescence intensity. Therefore, theoretically, even if the copy number is 1 copy / ml, the virus copy number can be quantified.

  The term “having hydrophilicity” includes not only the case where the inner surface of the well is subjected to hydrophilic treatment such as plasma treatment, but also the case where the material itself has hydrophilicity. At least, the hydrophilicity of the inner surface of the well should be high with respect to the surface of the well plate (excluding the well).

  The surface of the well plate is hydrophobic, and the nucleic acid extract is drawn into the well by wetting one side of the well plate with the nucleic acid extract, and excess nucleic acid extract is removed from the well plate. After removing from the top, it is desirable to enclose the nucleic acid extract in the well by covering both surfaces of the well plate with lid members.

  In this way, after drawing the nucleic acid extract into the well, both sides of the well are sealed, so that the nucleic acid extract can be surely filled even in a minute well, and entrapment of air or the like is prevented. Can do.

  The well plate is provided with a plurality of well groups composed of a plurality of wells of the same size, and the plurality of well groups have different well sizes, and each well group has a predetermined amount of sample liquid per sample solution. The number of virus copies can be counted.

  In this way, by forming multiple well groups consisting of wells of different sizes on a single well plate or multiple well plates, the measurement range can be expanded from low to high concentration samples. It can be accurately detected and quantified.

  Although details will be described later, for example, when a well plate having a total well capacity of 1 ml is used, the copy number of the well is 1 copy / ml (in this case, the number of wells where fluorescence is generated (disappears) becomes 1). Can be quantified. However, in order to quantify the copy number to 1 million copies / ml using this well plate, the number of wells of 1 million or more is required. This increases the manufacturing cost of the well plate and increases the size of the well plate. On the other hand, in the case of a well plate having a total well volume of 0.01 ml, the lower limit of the copy number measurement is 100 copies / ml. However, if the number of wells is 10,000 or more, the quantity is quantified to 1 million copies / ml. Can be

  Thus, there are appropriate well sizes and quantities depending on the measurement range depending on the nucleic acid concentration of the virus. Therefore, by providing a plurality of well groups having different sizes on one well plate, a wide range of virus concentrations can be measured with one well plate, and the well plate can be miniaturized.

  A plurality of well groups having different well sizes are provided on different well plates, and the number of virus copies per predetermined amount of the sample liquid can be counted for each well group of the different well plates.

  By using a plurality of well plates each having a different well group, the number of virus copies per predetermined amount of the sample liquid can be counted for each well group.

  Even when a plurality of well plates having well groups of different sizes are used, it is possible to accurately detect and quantify samples having a low virus concentration to samples having a high virus concentration. In this case, a plurality of well plates may be installed in the apparatus at the same time, and a PCR reaction may be performed at the same time for analysis, or a PCR reaction may be performed at different timings for analysis. That is, a well plate having a plurality of well groups having different sizes at the same time in a single virus test may be used, and each well plate may be divided into a plurality of tests. Note that it is desirable to use a well plate having a plurality of well groups having different sizes at a time because of higher efficiency.

  The nucleic acid extract contains an internal standard, and the number of the wells in which the fluorescence of the internal standard is generated or the number of lost wells is counted, and when a predetermined number or more of the wells are counted, a PCR reaction It is desirable to judge that is effective.

  In this way, the effectiveness of the PCR reaction can be determined. For this reason, it is possible to prevent making an erroneous determination when, for example, a trouble occurs in the device or the mixing of the solution.

  A second aspect of the present invention is a virus test apparatus, wherein a well plate having a plurality of wells whose inner surfaces are hydrophilic is used, the temperature control unit on which the well plate is mounted and the temperature of the well plate can be controlled; A light source that irradiates light to the well, a detection unit that detects fluorescence in the well, and the number of wells in which fluorescence has been detected or the number of wells in which fluorescence has been detected detected by the detection unit And the temperature controller performs a predetermined number of PCR reactions on the nucleic acid extract in the well, and the analysis unit detects the detection when the well is irradiated with a light source. A virus test apparatus for calculating the number of copies of virus per predetermined amount of a sample liquid from the number of fluorescence-generating wells detected or fluorescence-depleted wells and the total volume of the wells That.

  By using this apparatus, as described above, it is possible to accurately quantify from a low concentration to a high virus concentration. In particular, in the case of a low virus concentration, the present invention is particularly important because it cannot be measured by a conventional method of measuring fluorescence intensity. In the present invention, since the PCR reaction is performed in a large number of wells having a small pore size, the volume of the liquid in each well is small. Time is shortened, the inspection time is shortened, and the apparatus can be miniaturized.

  3rd invention is a well plate used for a virus test | inspection, Comprising: The inner surface of a well has several wells which are hydrophilic, The surface except the said well is hydrophobic, The said well plate has the same size A well plate comprising at least one well group comprising a plurality of the wells. Further, a well plate used for a virus test, the inner surface of the well has a plurality of wells subjected to hydrophilic treatment, the surface excluding the well is hydrophobic, and the well plate has the same size. A well plate comprising a plurality of well groups each including a plurality of wells, wherein the well groups having different well sizes are formed on the same well plate.

  According to the third invention, a sample having a wide range of virus concentrations is contained in a well plate using a plurality of well plates or a well plate having a plurality of well groups having different sizes on one well plate. The number of copies of the sample DNA can be quantified.

  ADVANTAGE OF THE INVENTION According to this invention, even if it is a low concentration virus, it can measure accurately and can provide the virus inspection method etc. which are excellent also in reproducibility.

The figure which shows the well plate 3. FIG. (A)-(c) is a figure which shows the process of filling each well 5 of the well plate 3 with a nucleic acid extract. (A)-(c) is a figure which shows the process of filling each well 5 of the well plate 3 with a nucleic acid extract. Sectional drawing which shows the fitting state of the application | coating jig 25 at the front-end | tip of the pipette 23. FIG. The figure which shows the state which fills a well 5 with a nucleic acid extract with the pipette 23 to which the application | coating jig | tool 25 was attached. The figure which shows the virus inspection apparatus. The figure which shows the virus inspection apparatus. (A) is a figure which shows the fluorescence emission well 8a and the fluorescence non-emission well 8b by an internal standard, (b) is a figure which shows the fluorescence emission well 6a and the fluorescence non-emission well 6b by a virus. The figure which shows the well plate 3a which consists of a some well group of a mutually different well size. The conceptual diagram which shows the change of the conventional PCR cycle and fluorescence intensity.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description, fluorescence-enhanced PCR will be described.

  First, a virus is dissolved from serum to extract nucleic acid. For example, magnetic particles are used for nucleic acid extraction. After sufficiently reacting the magnetic particles with the nucleic acid, the magnetic particles are collected using a magnet. After washing the collected magnetic particles, the nucleic acids are collected by dissolving in a buffer solution that separates the magnetic particles and the nucleic acid.

  Next, a constant concentration of internal standard and PCR reagents (hairpin primer, fluorescent indicator, etc.) are added to the obtained nucleic acid extraction solution. The internal standard is, for example, a PUC18 plasmid, which is amplified by a PCR reaction. Further, as the fluorescent indicator, for example, a fluorescent increase indicator is used. Here, when the DNA of the nucleic acid extraction solution is detected with the fluorescence-increasing indicator, the fluorescence-increasing indicator is used as the internal standard. When the fluorescence wavelength is 450 nm or less, the influence of self-emission of the plastic becomes large. Therefore, the fluorescence wavelength is preferably 500 nm or more. In addition, instead of the fluorescence increase indicator, a fluorescence decay indicator can be used. In this case, the DNA of the nucleic acid extraction solution is detected with the fluorescence decay indicator.

  Next, a specimen (nucleic acid extraction solution) is applied on the well plate. FIG. 1 is a view showing a well plate 3. The well plate 3 is provided with a plurality of wells 5. The well plate 3 is made of resin, glass or metal. The well plate 3 can be manufactured by any known method such as injection, etching, pressing, laser processing, and printing, although the manufacturing method differs depending on the material.

  FIG. 2A is a partial cross-sectional view of the well plate 3. As shown in FIG. 2A, the well 5 penetrates in the thickness direction of the well plate 3. A hydrophilic treatment 7 is applied to the inner surface of the well 5. As the hydrophilic treatment 7, for example, there is a method of modifying the surface of the resin. As surface modification methods, surface modification methods such as UV irradiation, corona discharge, plasma treatment, and urea treatment are known, and these methods can be used.

  When the well plate 3 is made of glass, hydrophilic treatment is unnecessary because the inner surface of the well 5 is hydrophilic due to the hydrophilicity of the glass itself. Further, both surfaces of the well plate 3 (surfaces excluding the inner surface of the well 5) are subjected to hydrophobic treatment. That is, the inner surface of the well 5 has higher hydrophilicity than both surfaces of the well plate 3.

  Next, as shown in FIG. 2B, the nucleic acid extract 9 is applied to one surface of the well plate 3. Note that one surface of the well plate 3 may be wetted with the nucleic acid extract 9 by a method other than the illustrated coating method. At this time, the nucleic acid extract 9 contains a certain concentration of nucleic acid 11. As described above, since the inner surface of the well 5 is hydrophilic, the nucleic acid extract 9 is drawn into the well 5 (arrow A in the figure).

  By doing so, as shown in FIG. 2C, a certain amount of the nucleic acid extract 9 is dispensed inside each well 5. At this time, the nucleic acid 11 enters into some of the wells 5 according to the concentration. Note that it is not necessary for all the nucleic acid extract 9 (including the nucleic acid 11) to be drawn into the well 5.

  Next, as shown in FIG. 3A, the excess nucleic acid extract 9 on the well plate 3 that has not entered the well 5 is removed. At this time, since the surface of the well plate 3 is hydrophobic, the nucleic acid extract 9 can be easily removed.

  Also, the pipette 23 and the application jig 25 as shown in FIG. 4 can be used for filling the well with the nucleic acid extract 9 and removing excess nucleic acid extract on the outer surface of the well. The application jig 25 is used by being fitted to the tip of the pipette 23. A hole 27 communicating with the pipette 23 is provided at the tip of the application jig 25. Therefore, when the application jig 25 is fitted with the nucleic acid extract 9 sucked into the pipette 23, the nucleic acid extract 9 can be discharged from the tip of the application jig 25. In addition, the hole 27 is expanded in diameter as it goes to the tip.

  As shown in FIG. 5, with the application jig 25 fitted to the pipette 23, the nucleic acid is continuously extracted into the well 5 by sweeping it horizontally in a predetermined direction at a distance of several tens of μm from the well plate 3. The liquid can be dispensed. At this time, if the area of the hole 27 at the tip of the application jig 25 can be set to a size that covers a predetermined number of wells 5 on the well plate, the nucleic acid extract 9 can be efficiently applied to the wells 5. Can be done manually.

  The excess nucleic acid extract 9 that has not entered the well 5 can be removed by sweeping the upper surface of the well plate 3 with a blade. Here, the removal of the coating solution with the blade may be provided in the coating step with the blade, but a blade matching the coating width of the nucleic acid extraction solution 9 is provided on the outer periphery of the coating jig 25 to apply the nucleic acid extraction solution 9. And the excess nucleic acid extract 9 can be removed simultaneously.

  After removing the excess nucleic acid extract 9 on the well plate 3, as shown in FIG. 3B, a lid member 13a is provided on one surface thereof. Moreover, as shown in FIG.3 (c), the well plate 3 is turned over and the cover member 13b is provided in the other surface. By doing so, the nucleic acid extract 9 is enclosed in the well 5. Here, when the nucleic acid extract 9 protruding from the well after inversion is swept, it is desirable to sweep the upper surface of the well plate with the blade as described above.

  As described above, the nucleic acid extract 9 can be injected into each of the wells 3 having a group of wells having different pore diameters by the above-described method.

  In addition to the above, the method of providing the lid member on the well plate 3 includes injecting the nucleic acid extract into the well plate 3 and then closing the lower surface of the well plate 3 with a hydrophobic tape provided on the surface of the lid member. After the nucleic acid extract 9 pushed out on the upper surface of the plate 3 is swept away with a blade, a lid member can be attached to the upper part.

  Next, the well plate 3 into which the nucleic acid extract has been dispensed is placed on a temperature controller. FIG. 6 is a schematic diagram showing the virus inspection apparatus 1. On the temperature controller 19, a plurality of well plates 3 in which specimens are placed are placed. In some cases, a well plate 3 in which a sample solution dispensed from a nucleic acid of a known concentration with a different concentration is injected into a well as an external standard is also placed at the same time. Because of its excellent detection sensitivity, an external standard is usually not used. A pressurizing unit 20 is disposed on the upper portion of the well plate 3. A light source 15 and cameras 17a and 17b are arranged above the temperature controller 19 on which the well plate 3 is placed.

The pressure unit 20 is heated to 100 ° C., for example. The pressurizing unit 20 pressurizes the well plate 3 or generates an internal force (internal pressure) on the well plate 3. For example, each well 5 is configured to pressurize at about 1 to 1.5 kg / cm ² or generate an internal force (internal pressure). In the case of a method of generating an internal force (internal pressure), a structure in which a plate member such as glass having high rigidity is placed above and below the well plate and the well plate is sandwiched from above and below the plate member can be considered. For this reason, in any case, the lid members 13 a and 13 b are pressed against the main body of the well plate 3. By doing in this way, the nucleic acid extract 9 is not mixed between the adjacent wells 5 in the PCR reaction.

  In this state, the temperature controller 19 sequentially changes the temperature of the nucleic acid extract 9 in each well 5 at a predetermined temperature cycle, for example, the temperature at which the nucleic acid is amplified (55 ° C. to 95 ° C.), A PCR reaction is performed. Thus, the nucleic acid can be amplified.

  When the predetermined number of PCR reactions are completed, the pressurizing unit 20 is removed and light of a predetermined wavelength is irradiated from the light source 15 as shown in FIG. For example, a halogen lamp is used as the light source 15. However, the light source is not limited to the halogen lamp as long as the intensity is high enough to excite fluorescence, and a laser can also be used. Here, when a laser is used as the light source, the wavelength of the laser used differs between the internal standard excitation laser and the fluorescence excitation laser of the sample liquid. The light source 15 irradiates the entire surface of the well plate 3 (all wells 5) with a mirror (not shown). Note that the lid member 13a (13b) on the light source 15 side is made of an amorphous resin such as polycarbonate that is transparent to transmit light and fluorescence emitted from the light source, and thus the lid member 13a (13b). ) Is closed and measurement is possible. Further, by forming a reflective film on the surface of the lid member 13b (13a) opposite to the light source 15 facing the well 5, light leakage can be prevented and fluorescence can be measured efficiently from above.

  Here, when testing the nucleic acid extract, when adjusting the nucleic acid extract, adjust so that viral DNA of known concentration is added to all wells as an internal standard, and amplify the DNA to investigate the copy number of the DNA. Add to.

  Here, in the present invention, the number of copies of DNA in the internal standard of each well and the nucleic acid extract to be tested is different from the fluorescence wavelength of the fluorescent dye that emits light after each PCR reaction. Use to find. For example, in the camera 17a, the number of copies of DNA is obtained by measuring the fluorescence using a filter in which the number of fluorescent light emitting wells of the internal standard or the number of wells of the non-fluorescent light emitting well is matched with the fluorescent wavelength of the internal standard, and processing this image. Ask for. In the camera 17b, fluorescence is measured using a filter in which the number of fluorescent light-emitting wells or the number of wells in the non-fluorescent light-emitting well in the nucleic acid extract to be examined is matched with the fluorescence emission wavelength of the nucleic acid extract, and this is subjected to image processing. To determine the DNA copy number.

  Here, the fluorescence wavelength used for the measurement of the internal standard and the fluorescence wavelength used for the detection of DNA in the nucleic acid extract are different from each other so that the fluorescence results of the mutual fluorescent substances do not appear in the measurement results. Different fluorescent materials shown are used. For this reason, the fluorescence length detected by the camera 17a and the fluorescence wavelength detected by the camera 17b are naturally different.

  Here, first, the camera 17a (FIG. 7) that measures the internal standard fluorescent light emitting well and the non-fluorescent light emitting well images all the wells 5. That is, the fluorescent light emitting well 8a or the fluorescent non-light emitting well 8b is detected by the camera 17a. Further, an analysis unit (not shown) connected to the camera 17a counts the number of wells of the fluorescent light emitting well 8a or the fluorescent non-light emitting well 8b according to the internal standard for each well 5. The analysis unit determines that the PCR reaction is appropriate when a certain number or more of the fluorescence emission wells 8a exist. When a fluorescence decay type PCR reaction is performed, it is determined that the PCR reaction is appropriate when a certain number or more of the non-luminescent wells 8b exist.

  For example, FIG. 8A is a conceptual diagram showing a part of the well plate 3 showing a fluorescent light emitting well 8a in which fluorescence of a specific wavelength is generated by an internal standard and a fluorescent non-light emitting well 8b in which no fluorescence is generated. is there. The well 5 irradiated with the light source is divided into a well that generates fluorescence and a well that does not generate fluorescence. As shown in the figure, in some of the wells 5, fluorescence may not be generated. This is the case, for example, when the PCR reaction is not properly performed.

  The material of the well plate 3 is made of resin, glass or metal. However, when using a resin material or glass material, it is desirable to select a resin material or glass material having low light transmittance. Even if the fluorescence in each well 5 is transmitted to the adjacent well 5, it is attenuated or absorbed in the well plate, and is emitted out of the well plate by repeating total reflection. be able to. In any case, in the present invention, the fluorescence intensity of each well is not quantitatively measured, and it is only necessary to distinguish whether each well is a fluorescent light emitting well or a non-light emitting well. It is not necessary to add.

  FIG. 8B shows a well plate 3 showing a fluorescent light emitting well 6a in which fluorescence of a specific wavelength is generated by a virus (nucleic acid) to be measured by a sample liquid and a fluorescent non-light emitting well 6b in which no fluorescence is generated. It is a conceptual diagram which shows a part. The fluorescent light emitting well 6a is a well in which nucleic acid was present when the nucleic acid extract was dispensed into the well 5. When one nucleic acid is present in the well 5, the nucleic acid is amplified by the PCR reaction. Therefore, as this reaction proceeds, fluorescence is amplified.

  Measure fluorescent wells and non-fluorescent wells of DNA in the nucleic acid extract to be examined. The camera 17b (FIG. 7) images all the wells 5. That is, the fluorescent light emitting well 6a or the fluorescent non-light emitting well 6b is detected by the camera 17b. Further, in the analysis unit (not shown) connected to the camera 17b, for each well 5, a well having a predetermined fluorescence intensity after a predetermined PCR cycle is recognized as a fluorescence emission well 6a. The analysis unit counts the number of wells of the fluorescent light emitting well 6a or the fluorescent non-light emitting well 6b due to the nucleic acid in the virus to be measured, and calculates the number of wells in which the nucleic acid originally existed.

  Here, if the concentration of the nucleic acid 11 relative to the capacity of one well 5 is sufficiently small and the concentration is completely uniform, the number of the nucleic acids 11 present in each well 5 is 1 or 0. Therefore, the number of fluorescent light-emitting wells 6a is the number of nucleic acids 11 contained in the initial predetermined amount of nucleic acid extract 9. Therefore, the analysis unit can calculate the number of copies per predetermined amount of the nucleic acid in the nucleic acid extract by dividing the number of the fluorescent light emitting wells 6a by the total capacity of the well 5.

  For example, when the well plate 3 having 50,000 wells 5 having a depth of 160 μm × 1000 μm is used, the total capacity of the wells 5 is about 1 ml. Therefore, for example, if the number of fluorescent light emitting wells 6a is 2000, the number of virus copies in the sample can be calculated as 2000 copies / ml. In addition, if such a well plate is used, the range of copy number 1-50,000 copies / ml can be measured correctly theoretically.

  When measuring a specimen having a larger copy number, the number of wells 5 described above may be increased. However, if the number of wells 5 is increased, the well plate 3 becomes larger. For example, in the above-described example, if the number of wells 5 is 50,000, the well plate 3 of about 50 mm square may be used. However, if one million wells 5 are to be formed, an area 20 times larger than that is required. Further, since the number of wells 5 processed increases, the manufacturing cost increases.

  In this case, the size of each well 5 may be reduced. However, there is a limit to the sensitivity of fluorescence measurement to reduce the well diameter, and the lower limit is 30 μm. For example, when the well plate 3 having 200,000 wells 5 having a depth of 80 μm × 100 μm is used, the total capacity of the wells 5 is about 0.1 ml. Therefore, for example, if the number of fluorescent light emitting wells 6a is 100,000, the number of virus copies in the sample can be calculated as 1 million copies / ml. That is, when one fluorescent light emitting well 6a exists in 0.1 ml, the number of copies per ml is calculated as 10. Therefore, if such a well plate 3 is used, theoretically, it is possible to accurately measure the copy number range of 10 to 2 million copies / ml. Even in this case, since the size of the well plate 3 may be about 60 mm square, it does not increase in size.

  Further, when the well plate 3 having 200,000 wells 5 having a depth of Φ40 μm × 40 μm is used, the total capacity of the wells 5 is about 0.01 ml. Therefore, for example, if the number of fluorescent light emitting wells 6a is 100,000, the number of virus copies in the sample can be calculated as 10 million copies / ml. In addition, if such a well plate 3 is used, theoretically, the range of the copy number of 100 to 20 million copies / ml can be accurately measured. Even in this case, since the size of the well plate 3 may be about 35 mm square, it does not increase in size.

  Thus, the range that can be measured accurately varies depending on the size (capacity) and the number of wells 5 on the well plate 3. As described above, in the present invention, theoretically, it is possible to accurately measure from a copy number of 1 copy / ml. The size of the well 5 is preferably about Φ35 to 200 μm, for example, and the depth is preferably 80 to 1000 μm.

  As shown in FIG. 9, a well plate 3a composed of a plurality of well groups 21a and 21b can be used. The well groups 21a and 21b are each composed of wells 5 of the same size, but the size of the well 5a of the well group 21a is different from the size of the well 5b of the well group 21b. For example, suppose that the well group 21a is provided with 30,000 wells 5a each having a depth of Φ200 μm × 1000 μm. In addition, it is assumed that 10,000 wells 5b each having a depth of 35 μm × 1000 μm are provided in the well group 21b.

  In this case, the total volume of the well 5a of the well group 21a is about 1 ml. For this reason, it is possible to measure up to a copy number of 1 to 30,000 copies / ml by the well group 21a. On the other hand, in the well group 21b, the total volume of the well 5b is about 0.01 ml. For this reason, it is possible to measure the number of copies from 1 to 1 million copies / ml by the well group 21b. By doing in this way, it is possible to measure from 1 to 1 million copies / ml with one well plate 3a.

  In this case, it is possible to measure with high accuracy over a wide range by setting the size of the well group so that a plurality of measurement ranges overlap.

  Note that the well size of the well group is not limited to the above-described example. Further, the thickness of the well plate (depth of the well 5) may be changed for each well group. Further, three or more well groups may be provided on one well plate.

  As described above, according to the present embodiment, the number of nucleic acid copies in a virus can be calculated by counting the number of fluorescent light emitting wells 6a. Therefore, as in the conventional method, one sample is dispensed into one well 5, and the number of copies of the virus is measured with higher accuracy than when the original copy number is estimated based on the fluorescence intensity corresponding to the PCR cycle. can do.

  Theoretically, it can be measured from a copy number of 1 copy / ml. Therefore, it is possible to measure even low-concentration viruses that have been difficult to quantify in the past. In addition, the design of the well plate 3 enables the virus copy number to be accurately measured over a wide range from a low concentration to a high concentration.

  Further, since the inner surface of the well 5 is hydrophilic, the nucleic acid extract 9 can be easily dispensed into the well 5 by applying the nucleic acid extract 9 to one surface of the well plate 3 and wetting it. . Further, by making the surface of the well plate 3 hydrophobic, it is easy to remove the excess nucleic acid extract 9.

  Further, by pressurizing the well plate 3 during the PCR reaction using the pressurizing unit 20, it is possible to prevent the internal nucleic acid extract 9 from being mixed with other adjacent wells 5. At this time, since the pressure unit 20 is heated, the temperature in the well 5 is not taken away by the pressure unit, and the temperature in the well 5 can be made substantially uniform.

  Further, by forming a plurality of well groups 21a and 21b on one well plate 3a, it is possible to measure a wide range of virus concentrations from low concentration to high concentration with one well plate 3a.

  Thus, according to the present invention, it is possible to measure with high sensitivity even at a low concentration as compared with the conventional apparatus (lower limit sensitivity copy number 125 / ml). In addition, since only the fluorescence wells 6a after the PCR reaction are counted, conventionally, it took about 72 hours for the examination time. However, for example, from nucleic acid extraction to PCR, all of the time is about 2.5 hours. Can be completed. Moreover, since the well plate 3 is about 60 mm square, for example, the apparatus can be miniaturized.

  As mentioned above, although embodiment of this invention was described referring an accompanying drawing, the technical scope of this invention is not influenced by embodiment mentioned above. It is obvious for those skilled in the art that various modifications or modifications can be conceived within the scope of the technical idea described in the claims. It is understood that it belongs.

  For example, in the above-described example, the application to the hairpin primer PCR method has been described, but the present invention is not limited to this. It can be applied to other PCR methods. Further, as described above, for fluorescence decay type PCR, the virus concentration can be calculated by counting the number of fluorescence disappearances.

  In addition, when the copy number is large, the measurement can be performed up to a large copy number by using the same well plate by diluting the nucleic acid extract.

DESCRIPTION OF SYMBOLS 1 ......... Virus test | inspection apparatus 3, 3a ......... Well plates 5, 5a, 5b ......... Wells 6a, 8a ......... Fluorescence emission well 7 ......... Hydrophilic treatment 6b, 8b ......... Fluorescence non-emission well 9 ... Nucleic acid extract 11 ... Nucleic acid 13a, 13b ... Lid member 15 ... Light source 17a, 17b ... Camera 19 ... Temperature controller 20 ... Pressurization sections 21a, 21b ... … Well group 23 ………… pipette 25 ……… painting jig 27 ……… hole

Claims (9)

A virus inspection method,
Using a well plate having a plurality of wells whose inner surface is hydrophilic, filling the well with a nucleic acid extract,
With the well sealed, a predetermined number of PCR reactions are performed,
Count the number of wells in which fluorescence has occurred or the number of wells in which fluorescence has disappeared,
A virus inspection method characterized in that the number of copies of virus per predetermined amount of sample liquid is determined from the total volume of the well.   The surface of the well plate is hydrophobic, and the nucleic acid extract is drawn into the well by wetting one side of the well plate with the nucleic acid extract, and excess nucleic acid extract is removed from the well plate. 2. The virus testing method according to claim 1, wherein the nucleic acid extract is sealed in the well by covering both surfaces of the well plate with a lid member after removing from the top.   The well plate is provided with a plurality of well groups composed of a plurality of wells of the same size, and the plurality of well groups have different well sizes, and each well group has a predetermined amount of sample liquid per sample solution. 3. The virus inspection method according to claim 1, wherein the number of virus copies is counted.   A plurality of well groups having different well sizes are provided on the different well plates, and the number of virus copies per predetermined amount of the sample liquid is counted for each well group of the different well plates. The virus inspection method according to claim 1 or 2.   5. The number of virus copies per predetermined amount of sample liquid is counted for each well group by simultaneously using a plurality of well plates each having the well group with different well sizes. The virus inspection method described.   The nucleic acid extract contains an internal standard, and the number of the wells in which the fluorescence of the internal standard is generated or the number of lost wells is counted, and when a predetermined number or more of the wells are counted, a PCR reaction The virus inspection method according to claim 1, wherein it is determined that is effective. A virus inspection device,
Using a well plate having a plurality of wells whose inner surface is hydrophilic,
A temperature controller on which the well plate is mounted and capable of controlling the temperature of the well plate;
A light source for irradiating the well with light;
A detector for detecting fluorescence in the well;
An analysis unit that counts the number of wells in which fluorescence has been detected or the number of wells in which fluorescence has disappeared, detected by the detection unit;
Comprising
The temperature controller performs a predetermined number of PCR reactions on the nucleic acid extract in the well,
The analysis unit calculates the number of copies of virus per predetermined amount of the sample liquid from the number of fluorescence generation or fluorescence disappearance detected by the detection unit and the total volume of the well when the well is irradiated with a light source. A virus inspection apparatus characterized by: A well plate used for virus testing,
The inner surface of the well has a plurality of wells that are hydrophilic,
The surface excluding the well is hydrophobic,
The well plate is provided with at least one well group including a plurality of wells of the same size. A well plate used for virus testing,
The inner surface of the well has a plurality of wells that are hydrophilic,
The surface excluding the well is hydrophobic,
The well plate is provided with a plurality of well groups composed of a plurality of wells of the same size,
A well plate in which the well groups having different well sizes are formed in the same well plate.

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